Design, cloning and expression of humanized monoclonal antibodies against human interleukin-5

ABSTRACT

A monoclonal antibody is provided which specifically binds to human interleukin-5. Also provided are a hybridoma which produces the monoclonal antibody; complementary DNAs which encode the heavy and light chain variable regions of the monoclonal antibody and CDRs therefrom; humanized monoclonal antibodies; and pharmaceutical compositions comprising the monoclonal antibody or anti-idiotypic antibodies directed against it, humanized monoclonal antibodies, binding fragments, binding compositions or single-chain binding proteins derived from the antibody and a physiologically acceptable carrier.

This application is a continuation of U.S. application Ser. No.08/284,516, filed Aug. 4, 1994, now U.S. Pat. No. 6,056,957, which isthe national stage of international Application No. PCT/US93/00759,filed Feb. 4, 1993, which is a continuation-in-part of U.S. applicationSer. No. 07/832,842, filed Feb. 6, 1992, now abandoned.

The invention relates to nucleic acids which encode the heavy and lightchain variable regions of a monoclonal antibody against humaninterleukin-5 and complementarity determining regions therefrom, and tohumanized antibodies and binding proteins based upon the monoclonalantibody.

BACKGROUND OF THE INVENTION

Interleukin-5 (IL-5) is a lymphokine secreted by activated T cells whichis biologically active on B cells and eosinophils. Because IL-5 replacesT lymphocytes in in vitro antibody responses to thymus-dependentantigens, it was formerly called T cell replacing factor [TRF; Dutton etal., Prog. Immunol. 1:355 (1971); Schimpl et al., Nature 237:15 (1972)].Because it also stimulates differentiation of B lymphocytes into IgM andIgG plaque-forming cells and the growth of B cell lymphomas in vitro, ithas also been called B cell growth factor II [BCGFII; Takatsu et al., JImmunol. 124:2414 (1980)].

Murine IL-5 consists of 133 amino acid residues, including a signalsequence of 20 residues and three potential N-glycosylation sites.Deglycosylation does not affect the biological activity of murine IL-5in a B cell proliferation assay [Tavernier et al., DNA 8:491 (1989)].Human IL-5 consists of 134 amino acid residues, including a signalsequence of 19 residues and two potential N-glycosylation sites. Thestructures of both proteins have been described by Yokota e t al. [Proc.Natl. Acad. Sci. USA 84:7388 (1987)] and Kinashi et al. [Nature 324:70(1986)]. The degrees of homology of murine and human IL-5 at thenucleotide and amino acid sequence level are 77 and 70%, respectively.

Both murine and human IL-5 exist as homodimers linked by disulfidebonds. Therefore, glycosylated recombinant human IL-5 migrates in SDSpolyacrylamide gel electrophoresis with an apparent molecular weight of40,000 daltons under non-reducing conditions, and 20-22,000 daltonsunder reducing conditions [Tsujimoto et al., J. Biochem. 106:23 (1989)].

The cloning and expression of murine IL-5 has been described, e.g., byKinashi et al. [Nature 324:70 (1986)] and Takatsu et al. [J. Immunol.134:382 (1985)]. Human IL-5 complementary DNA (cDNA) has been isolatedusing murine IL-s cDNA as a probe by Azuma et al. [Nucleic Acids Res.14:9149 (1986)].

IL-5 has been shown to act as a maintenance and differentiation factorfor eosinophils. In humans, the activity of IL-5 appears to be specific,affecting eosinophils primarily. Human IL-5 induces eosinophil precursorcells to become mature cells. Moreover, the survival of eosinophilsisolated from circulating blood can be prolonged when human IL-5 ispresent in the culture media. Human IL-5 also stimulates culturedeosinophils to degranulate, and to release toxic proteins such as majorbasic protein (MBP) and eosinophil-derived neurotoxin (EDN) [Kita etal., J. Immunol. 149:629 (1992)].

It has been suggested that eosinophils kill parasites followinginfection and also play a significant role in inflammatory and allergicdiseases [see, e.g., Sanderson, Blood 79:3101 (1992)]. Increased levelsof eosinophils among circulating leukocytes have been observed followingparasitic infections and in certain chronic inflammatory tissues, suchas in asthmatic alveoli. Eosinophil infiltration and toxic granulerelease from eosinophils may play a role in tissue destruction and mayaggravate the symptoms of asthma.

For example, Gleich et al. [Adv. Immunol. 39:177 (1986)] and Frigas etal. [J. Allergy Clin. Immunol. 77:527 (1986)] have shown thathigh-density eosinophils and eosinophil major basic protein (MBP) areassociated with bronchial asthma and related tissue damage.

Recently, Coffman et al. (International Patent Application PublicationNo. WO/04979) have shown that antibodies against IL-5 can prevent orreduce eosinophilia which is associated with certain allergic diseasessuch as asthma. Monoclonal antibodies which specifically bind to andneutralize the biological activity of human IL-5 can be used for thispurpose.

A monoclonal antibody against IL-5 has been reported to have a prominenteffect in reversing parasite-induced eosinophilia in experimentalanimals [Schumacher et al, J. Immunol. 141:1576 (1988); Coffman et al.,Science 245:308 (1989)], suggesting that neutralizing antibodies may beclinically useful in relieving eosinophilia-related symptoms byantagonizing IL-5. In fact, it has been reported that when rodents ormonkeys bearing experimentally induced eosinophilia were treated withTRFK 5, a rat anti-mouse IL-5 monoclonal antibody, eosinophil counts inboth circulation and bronchial lavage were found to return to normallevels. Thus, neutralizing monoclonal antibodies may be effectiveantagonists.

Because most monoclonal antibodies are of rodent origin, however, thereis an increased likelihood that they would be immunogenic if usedtherapeutically in a human being, particularly over a long period oftime. To reduce this possibility, there is a need for recombinant or“humanized” antibodies against human IL-5. Such antibodies could be usedfor the treatment of conditions associated with eosinophilia, or for thetreatment of any other condition attributable to the biological activityof IL-5.

Initial efforts to reduce the immunogenicity of rodent antibodiesinvolved the production of chimeric antibodies, in which mouse variableregions were fused with human constant regions [Liu et al., Proc. Natl.Acad. Sci. USA 84:3439 (1987)]. It has been shown, however, that miceinjected with hybrids of human variable regions and mouse constantregions develop a strong anti-antibody response directed against thehuman variable region. This suggests that in the human system, retentionof the entire rodent Fv region in such chimeric antibodies may stillgive rise to human anti-mouse antibodies.

It is generally believed that CDR loops of variable domains comprise thebinding site of antibody molecules, the grafting of rodent CDR loopsonto human frameworks (i.e., humanization) was attempted to furtherminimize rodent sequences [Jones et al., Nature 321:522 (1986);Verhoeyen et al., Science 239:1534 (1988)]. Studies by Kabat et al. [J.Immunol. 147:1709 (1991)] have shown that framework residues of antibodyvariable domains are involved in CDR loop support. It has also beenfound that changes in framework support residues in humanized antibodiesmay be required to preserve antigen binding affinity. The use of CDRgrafting and framework residue preservation in a number of humanizedantibody constructs has been reported, e.g., by Queen et al. [Proc.Natl. Acad. Sci. USA 86:10029 (1989)], Gorman et al. [Proc. Natl. Acad.Sci. USA 88:4181 (1991)] and Hodgson [Bio/Technology 9:421 (1991)].Exact sequence information has been reported for only a few humanizedconstructs.

Although a high degree of sequence identity between human and animalantibodies has been known to be important in selecting human antibodysequences for humanization, most prior studies have used a differenthuman sequence for animal light and heavy variable sequences. Sequencesof known antibodies have been used or, more typically, those ofantibodies having known X-ray structures, antibodies NEW and KOL. See,e.g., Jones et al., supra; Verhoeyen et al., supra; and Gorman et al.,supra.

Methods for engineering antibodies have been described, e.g., by Boss etal. (U.S. Pat. No. 4,816,397), Cabilly et al. (U.S. Pat. No. 4,816,567),Law et al. (European Patent Application Publication No. 438 310) andWinter (European Patent Application Publication No. 239 400).

Reliance on the relatively few antibodies for which X-ray structureshave been determined has led to the frequent use of different humanlight and heavy chain sequences from different antibodies, becausealthough only two human Fab crystal structures are known, several humanlight chain crystal structures have been determined. Such an approachmay require changing framework residues in the human heavy and lightchains to ensure correct chain association and, therefore, limits theapplicability of humanization.

There thus is a need for improved methods for making humanizedantibodies that are not based upon the relatively few knowncrystallographic structures.

SUMMARY OF THE INVENTION

The present invention fulfills the foregoing needs by providing novelmethods for the design of humanized antibodies, and specific antibodyantagonists of human IL-5 and pharmaceutical compositions containing thesame.

More particularly, this invention provides a method for selecting humanantibody sequences to be used as human frameworks for humanization of ananimal antibody comprising:

(a) comparing the heavy and light chain variable region sequences of ananimal monoclonal antibody that is to be humanized withoptimally-aligned sequences of the heavy and light chain variableregions of human antibodies for which sequence information is available,thereby determining the percent identities for each of the comparedsequences;

(b) determining the number of ambiguities in each of such human antibodysequences;

(c) comparing Pin-region spacing of the animal antibody sequences with(i) that of each of such human antibody sequences and with (ii) those ofother antibodies which have known 3-dimensional structures; and

(d) selecting the human antibody sequence which has the best combinationof:

(i) low number of sequence ambiguities, and

(ii) high percent identities and similar Pin-region spacing, based oncomparison to the animal antibody sequences.

This invention further provides a method for determining which variableregion residues of an animal monoclonal antibody should be selected forhumanization comprising:

(a) determining potential minimal and maximal residues of the animalmonoclonal antibody, wherein:

(i) such minimal residues comprise CDR structural loops plus residuesrequired to support and/or orient the CDR structural loops, and

(ii) such maximal residues comprise Kabat CDRs plus CDR structural loopsplus residues required to support and/or orient the CDR structural loopsplus residues which fall within about 10 Å of a CDR structural loop andpossess a water solvent accessible surface of about 5 Å² or greater;

(b) performing computer modeling of:

(i) a sequence of an animal monoclonal antibody which is to behumanized,

(ii) a human antibody framework sequence, and

(iii) all possible recombinant antibodies comprising the human antibodyframework sequence into which the minimal and maximal residues of step(a) have been inserted,

which computer modeling is performed using software suitable for proteinmodeling and structural information from a structurally-characterizedantibody that has a sequence most nearly identical to that of theselected human antibody framework sequence;

(c) comparing results obtained in the computer modeling of step (b); and

(d) selecting the minimal or maximal residues which produce arecombinant antibody having a computer-modeled structure closest to thatof the animal monoclonal antibody.

Preferably, the human antibody framework sequence is selected asdescribed above.

The present invention still further provides a monoclonal antibodyproduced by a hybridoma having the identifying characteristics of a cellline deposited under American Type Culture Collection Accession No. ATCCHB 10959, and the hybridoma itself.

This invention still further provides polypeptides comprising a heavy orlight chain variable region of a monoclonal antibody which have aminoacid sequences defined by SEQ ID NO: 1 and SEQ ID NO: 2, complementaritydetermining regions (CDRs) from such variable regions, and isolated DNAsencoding such variable regions and CDRs. These DNAs can be used toconstruct binding compositions, single-chain binding proteins,polypeptides which contain one or more of the CDRs and retain antigenbinding activity, and recombinant antibodies comprising such CDRs, allof which are a part of this invention.

This invention still further provides pharmaceutical compositionscomprising such monoclonal antibody or recombinant antibodies, bindingcompositions, single-chain binding proteins and polypeptides; and aphysiologically acceptable carrier.

BRIEF DESCRIPTION OF THE FIGURES

The present invention may be more readily understood by reference to thedescription and example below, and to the accompanying figures in which:

FIG. 1 is a schematic representation of plasmid pSRSMPA5H.

FIG. 2 is a schematic representation of plasmid pDSRGMPA5H.

FIG. 3 is a schematic representation of plasmid pDSRGMPA5L.

FIG. 4 is a schematic representation of plasmid pSRSMPA5L.

DESCRIPTION OF THE INVENTION

All references cited herein are hereby incorporated in their entirety byreference.

As used herein, the terms “DNA” and “DNAS” are defined as moleculescomprising deoxyribonucleotides linked in standard 5′ to 3′phosphodiester linkage, including both smaller oligodeoxyribonucleotidesand larger deoxyribonucleic acids.

Antibodies comprise an assembly of polypeptide chains linked together bydisulfide bridges. Two principal polypeptide chains, referred to as thelight chain and the heavy chain, make up all major structural classes(isotypes) of antibody. Both heavy chains and light chains are furtherdivided into sub regions referred to as variable regions and constantregions. Heavy chains comprise a single variable region and three orfour different constant regions, and light chains comprise a singlevariable region (different from that of the heavy chain) and a singleconstant region (different from those of the heavy chain). The variableregions of the heavy chain and light chain are responsible for theantibody's binding specificity.

As used herein, the term “CDR structural loops” means the three lightchain and the three heavy chain regions in the variable portion of anantibody that bridge β strands on the binding portion of the molecule.These loops have characteristic canonical structures [Chothia et al., J.Mol. Biol. 196:901 (1987); Chothia et al., J. Mol. Biol. 227:799(1992)].

The term “Kabat CDRs” refers to hypervariable antibody sequences onheavy and light chains as defined by Kabat et al. [Sequences of Proteinsof Immunological Interest, 4th Edition, 1987, U.S. Department of Healthand Human Services, National Institutes of Health].

As used herein, the term “heavy chain variable region” means apolypeptide which is from about 110 to 125 amino acid residues inlength, the amino acid sequence of which corresponds to that of a heavychain of a monoclonal antibody of the invention, starting from theamino-terminal (N-terminal) amino acid residue of the heavy chain.Likewise, the term “light chain variable region” means a polypeptidewhich is from about 95 to 130 amino acid residues in length, the aminoacid sequence of which corresponds to that of a light chain of amonoclonal antibody of the invention, starting from the N-terminal aminoacid residue of the light chain.

The terms Fab, Fc, F(ab)₂, and Fv are employed with their standardimmunological meanings [Klein, Immunology (John Wiley, New York, 1982);Parham, Chapter 14, in Weir, ed. Immunochemistry, 4th Ed. (BlackwellScientific Publishers, Oxford, 1986)].

As used herein the term “monoclonal antibody” refers to a homogeneouspopulation of immunoglobulins which are capable of specifically bindingto human IL-5. It is understood that human IL-5 may have one or moreantigenic determinants comprising (1) peptide antigenic determinantswhich consist of single peptide chains within human IL-5, (2)conformational antigenic determinants which consist of more than onespatially contiguous peptide chains whose respective amino acidsequences are located disjointedly along the human IL-5 polypeptidesequence; and (3) post-translational antigenic determinants whichconsist, either in whole or part, of molecular structures covalentlyattached to human IL-5 after translation, such as carbohydrate groups,or the like. The antibodies of the invention may be directed against oneor more of these determinants.

As used herein the term “binding composition” means a compositioncomprising two polypeptide chains (1) which, when operationallyassociated, assume a conformation having high binding affinity for humanIL-5, and (2) which are derived from a hybridoma producing monoclonalantibodies specific for human IL-5. The term “operationally associated”is meant to indicate that the two polypeptide chains can be positionedrelative to one another for binding by a variety of means, includingassociation in a native antibody fragment, such as Fab or Fv, or by wayof genetically engineered cysteine-containing peptide linkers or otherlinkers at the carboxyl termini.

Monoclonal antibodies can be prepared using standard methods, e.g., asdescribed by Kohler et al. [Nature 256:495 (1975); Eur. J. Immunol.6:511 (1976)]. Essentially, an animal is immunized by standard methodsto produce antibody-secreting somatic cells. These cells are thenremoved from the immunized animal for fusion to myeloma cells.

Somatic cells with the potential to produce antibodies, particularly Bcells, are suitable for fusion with a myeloma cell line. These somaticcells may be derived from the lymph nodes, spleens and peripheral bloodof primed animals. In the exemplary embodiment of this invention ratspleen cells are used, in part because these cells produce a relativelyhigh percentage of stable fusions with mouse myeloma lines. It would bepossible, however, to use human, mouse, rabbit, sheep or goat cells, orcells from other animal species instead.

Specialized myeloma cell lines have been developed from lymphocytictumors for use in hyridoma-producing fusion procedures [Kohler andMilstein, Eur. J. Immunol. 6:511 (1976); Shulman et al., Nature 276:269(1978); Volk et al., J. Virol. 42:220 (1982)]. These cell lines havebeen developed for at least three reasons. The first is to facilitatethe selection of fused hybridomas from unfused and similarlyindefinitely self-propagating myeloma cells. Usually, this isaccomplished by using myelomas with enzyme deficiencies that render themincapable of growing in certain selective media that support the growthof hybridomas. The second reason arises from the inherent ability oflymphocytic tumor cells to produce their own antibodies. The purpose ofusing monoclonal techniques is to obtain fused hybrid cell lines withunlimited life spans that produce the desired single antibody under thegenetic control of the somatic cell component of the hybridoma. Toeliminate the production of tumor cell antibodies by the hybridomas,myeloma cell lines incapable of producing endogenous light or heavyimmunoglobulin chains are used. A third reason for selection of thesecell lines is for their suitability and efficiency for fusion.

Many myeloma cell lines may be used for the production of fused cellhybrids, including, e.g., P3X63-Ag8, P3X63-AG8.653, P3/NS1-Ag4-1 (NS-1),Sp2/0-Agl4 and S194/5.XXO.Bu.1. The P3X63-Ag8 and NS-1 cell lines havebeen described by Kohler and Milstein [Eur. J. Immunol. 6:511 (1976)].Shulman et al. [Nature 276:269 (1978)] developed the Sp2/0-Agl4 myelomaline. The S194/5.XXO.Bu.1 line was reported by Trowbridge [J. Exp. Med.148:313 (1979)].

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually involve mixing somatic cells withmyeloma cells in a 10:1 proportion (although the proportion may varyfrom about 20:1 to about 1:1), respectively, in the presence of an agentor agents (chemical, viral or electrical) that promotes the fusion ofcell membranes. Fusion methods have been described by Kohler andMilstein, supra, Gefter et al. [Somatic Cell Genet. 3:231 (1977)], andVolk et al. (J. Virol. 42:220 (1982)]. The fusion-promoting agents usedby those investigators were Sendai virus and polyethylene glycol (PEG).

Because fusion procedures produce viable hybrids at very low frequency(e.g., when spleens are used as a source of somatic cells, only onehybrid is obtained for roughly every 1×10⁵ spleen cells), it isessential to have a means of selecting the fused cell hybrids from theremaining unfused cells, particularly the unfused myeloma cells. A meansof detecting the desired antibody-producing hybridomas among otherresulting fused cell hybrids is also necessary.

Generally, the selection of fused cell hybrids is accomplished byculturing the cells in media that support the growth of hybridomas butprevent the growth of the unfused myeloma cells, which normally would goon dividing indefinitely. The somatic cells used in the fusion do notmaintain long-term viability in in vitro culture and hence do not pose aproblem. In the example of the present invention, myeloma cells lackinghypoxanthine phosphoribosyl transferase (HPRT-negative) were used.Selection against these cells is made inhypoxanthine/aminopterin/thymidine (HAT) medium, a medium in which thefused cell hybrids survive due to the HPRT-positive genotype of thespleen cells. The use of myeloma cells with different geneticdeficiencies (drug sensitivities, etc.) that can be selected against inmedia supporting the growth of genotypically competent hybrids is alsopossible.

Several weeks are required to selectively culture the fused cellhybrids. Early in this time period, it is necessary to identify thosehybrids which produce the desired antibody, so that they maysubsequently be cloned and propagated. Generally, around 10% of thehybrids obtained produce the desired antibody, although a range of fromabout 1 to about 30% is not uncommon. The detection ofantibody-producing hybrids can be achieved by any one of severalstandard assay methods, including enzyme-linked immunoassay andradioimmunoassay techniques which have been described in the literature[see, e.g., Kennet et al. (editors), Monoclonal Antibodies andHybridomas: A New Dimension in Biological Analyses, pp. 376-384, PlenumPress, New York (1980)].

Once the desired fused cell hybrids have been selected and cloned intoindividual antibody-producing cell lines, each cell line may bepropagated in either of two standard ways. A suspension of the hybridomacells can be injected into a histocompatible animal. The injected animalwill then develop tumors that secrete the specific monoclonal antibodyproduced by the fused cell hybrid. The body fluids of the animal, suchas serum or ascites fluid, can be tapped to provide monoclonalantibodies in high concentration. Alternatively, the individual celllines may be propagated in vitro in laboratory culture vessels. Theculture medium containing high concentrations of a single specificmonoclonal antibody can be harvested by decantation, filtration orcentrifugation, and subsequently purified.

Monoclonal antibodies can also be produced using well known phagelibrary systems.

The use and generation of fragments of antibodies is well known, e.g.,Fab fragments [Tijssen, Practice and Theory of Enzyme Immunoassays(Elsevier, Amsterdam, 1985)], Fv fragments [Hochman et al., Biochemistry12:1130 (1973); Sharon et al., Biochemistry 15:1591 (1976); Ehrlich etal., U.S. Pat. No. 4,355,023] and antibody half molecules(Auditore-Hargreaves, U.S. Pat. No. 4,470,925). Moreover, such compoundsand compositions of the invention can be used to construct bi-specificantibodies by known techniques, e.g., by further fusions of hybridomas(i.e. to form so-called quadromas; Reading, U.S. Pat. No. 4,474,493) orby chemical reassociation of half molecules [Brennan et al., Science229:81 (1985)].

Hybridomas and monoclonal antibodies can be produced against human IL-5from any source, e.g., from commercial or natural sources or through theapplication of chemical synthetic methods or recombinant DNA technology.“Recombinant IL-5” is defined herein to mean IL-5 produced by expressionof recombinant DNA (cDNA) encoding the same in a prokaryotic oreukaryotic expression system. In addition, genomic DNA can be used forproducing IL-5 in eukaryotic systems. The IL-5 produced may beglycosylated or unglycosylated.

Since the nucleotide sequences of DNA encoding murine and human IL-5 areknown [see e.g., Azuma et al., Nucleic Acids Res. 14:9149 (1986)], suchDNAs can be chemically synthesized using the phosphoramidite solidsupport method of Matteucci et al. [J. Am. Chem. Soc. 103:3185 (1981)],the method of Yoo et al. [J. Biol. Chem. 764:17078 (1989)], or otherwell known methods. This can be done, for example, by synthesizingrelatively small oligonucleotides and ligating them together, analogousto the way that Barr et al. (International Patent ApplicationPublication No. WO 85/02200) chemically synthesized DNA encoding IL-2.

Alternatively, a cell line capable of making IL-5 can be stimulated tomake IL-5 mRNA, which can serve as a template to make IL-5 cDNA bystandard methods. A cDNA library can then be constructed in which IL-5cDNA can be identified using oligonucleotide probe mixtures based on theknown sequence information. This cDNA can then be cloned and expressedin one of the many available bacterial, yeast or mammalian expressionsystems. This method has been used to produce recombinant rat, mouse orhuman IL-5 [see, e.g., Tavernier et al., DNA 8:491 (1989); Minamitake etal., J. Biochem. 107:292 (1990); Uberla et al., Cytokine 3:72 (1991)].Human IL-5 was produced in a commonly-owned U.S. patent application(Ser. No. 07/615,061, filed Nov. 16, 1990) by expressing cDNA encodinghuman IL-5 in Chinese hamster ovary (CHO) cells. Tsujimoto et al. [J.Biochem. 106:23 (1989)] have also described the production ofrecombinant human IL-5 in CHO cells.

In still another approach, oligonucleotide probe mixtures based on knownIL-5 nucleotide sequences can be used to identify IL-5 genes in genomicDNA libraries prepared by standard methods. DNA thus identified can beexcised from the library by restriction endonuclease cleavage, sequencedand expressed in a eukaryotic expression system or (following introndeletion by standard methods if necessary) in a prokaryotic expressionsystem. In this way, Campbell et al. [Proc. Natl. Acad. Sci. USA 84:6629(1987)] produced human IL-5 in monkey kidney (COS) cells.

Of course, both cDNA and genomic DNA libraries can be screened by theapplication of standard expression cloning methods, instead of by theuse of oligonucleotide probes. IL-5 thus produced is detected throughthe use of known immunochemical or bioassay methods.

IL-5 polypeptides can also be made directly using synthetic peptidechemistry, e.g., as described by Merrifield [J. Am. Chem. Soc. 85:2149(1963)], or IL-5 can be purchased commercially.

Once a hybridoma producing the desired monoclonal antibody is obtained,techniques can be used to produce interspecific monoclonal antibodieswherein the binding region of one species is combined with a non-bindingregion of the antibody of another species [Liu et al., Proc. Natl. Acad.Sci. USA 84:3439 (1987)]. For example, the CDRs from a rodent monoclonalantibody can be grafted onto a human antibody, thereby “humanizing” therodent antibody [Riechmann et al., Nature 332:323 (1988)]. Moreparticularly, the CDRs can be grafted onto a human antibody variableregion with or without human constant regions. Such methodology has beenused, e.g., to humanize a mouse monoclonal antibody against the p55(Tac) subunit of the human interleukin-2 receptor [Queen et al., Proc.Natl. Acad. Sci. USA 86:10029 (1989)].

The cDNAs of the present invention which encode the heavy and lightchain variable regions of monoclonal antibodies specific for human IL-5and the CDRs from such antibodies can be engineered in such a fashion.The location of the CDRs within the variable regions of the antibodiescan be determined using a number of well known standard methods. Forexample, Kabat et al., supra, have published rules for locating CDRs.CDRs determined using these rules are referred to herein as “KabatCDRs.” Computer programs are also available which can be used toidentify CDR structural loops on the basis of the amino acid residuesinvolved in the three-dimensional binding site loops of the antibodychains, e.g., as described below.

The methods used herein are applicable to the humanization of a widevariety of animal antibodies. A two-step approach is used which involves(a) selecting human antibody sequences that are used as human frameworksfor humanization, and (b) determining which variable region residues ofan animal monoclonal antibody should be selected for insertion into thehuman framework chosen.

The first step involves selection of the best available human frameworksequences for which sequence information is available. This selectionprocess is based upon the following selection criteria:

(1) Percent Identities

The sequences of the heavy and light chain variable regions of an animalmonoclonal antibody that is to be humanized are optimally aligned andcompared preferably with all known human antibody heavy and light chainvariable region sequences. This is in contrast to the methods of theprior art, which rely heavily on the use of only two human antibodies,NEW and KOL. Structural information is available for these antibodies,the designations for which are the initials of human patients from whichthey were derived. The structure of antibody HIL is also known now(Brookhaven Code P8FAB).

Once the sequences are thus compared, residue identities are noted andpercent identities are determined. All other factors being equal, it isdesirable to select a human antibody which has the highest percentidentity with the animal antibody.

(2) Sequence Ambiguities

The known human antibody chain sequences are then evaluated for thepresence of unidentified residues and/or ambiguities, which are sequenceuncertainties. The most common of such uncertainties are mistakenidentification of an acidic amino acid for an amide amino acid due toloss of ammonia during the sequencing procedure, e.g., incorrectidentification of a glutamic acid residue, when the residue actuallypresent in the protein was a glutamine residue. Uncertainties areidentified by examination of data bases such as that of Kabat et al.,supra. All other factors being equal, it is desirable to select a humanantibody chain having as few such ambiguities as possible.

(3) Pin-region Spacing Antibody chain variable regions containintra-domain disulfide bridges. The distance (number of residues)between the cysteine residues comprising these bridges is referred to asthe Pin-region spacing [Chothia et al., J. Mol. Biol. 196:901 (1987)].All other factors being equal, it is most desirable that the Pin-regionspacing of a human antibody selected be similar or identical to that ofthe animal antibody. It is also desirable that the human sequencePin-region spacing be similar to that of a known antibody 3-dimensionalstructure, to facilitate computer modeling.

Based upon the foregoing criteria, the human antibody (or antibodies)having the best overall combination of desirable characteristics isselected as the framework for humanization of the animal antibody. Theheavy and light chains selected may be from the same or different humanantibodies.

The second step in the methods of this invention involves determinationof which of the animal antibody variable region sequences should beselected for grafting into the human framework. This selection processis based upon the following selection criteria:

(1) Residue Selection

Two types of potential variable region residues are evaluated in theanimal antibody sequences, the first of which are called “minimalresidues.” These minimal residues comprise CDR structural loops plus anyadditional residues required, as shown by computer modeling, to supportand/or orient the CDR structural loops.

The other type of potential variable region residues are referred to as“maximal residues.” They comprise the minimal residues plus Kabat CDRsplus any additional residues which, as determined by computer modeling,fall within about 10 Å of CDR structural loop residues and possess awater solvent accessible surface [Lee et al., J. Biol. Chem. 55:379(1971)] of about 5 Å² or greater. In the Example below, residues fallingwithin 5 Å of CDR structural loops were selected.

(2) Computer Modeling

To identify potential variable region residues, computer modeling iscarried out on (a) the variable region sequences of the animal antibodythat is to be humanized, (b) the selected human antibody frameworksequences, and (c) all possible recombinant antibodies comprising thehuman antibody framework sequences into which the various minimal andmaximal animal antibody residues have been grafted.

The computer modeling is performed using software suitable for proteinmodeling and structural information obtained from an antibody that (a)has variable region amino acid sequences most nearly identical to thoseof the animal antibody and (b) has a known 3-dimensional structure. Anexample of software that can be used is the SYBYL Biopolymer Modulesoftware (Tripos Associates). The antibody from which the structuralinformation can be obtained may be but need not necessarily be a humanantibody. For the Example below, structural information from a murineantibody designated 1F19 was used.

Based upon results obtained in the foregoing analysis, recombinantchains containing the animal variable regions producing a computermodeling structure most nearly approximating that of the animal antibodyare selected for humanization.

The nucleotide sequences of cDNAs encoding the partial heavy andcomplete light chain variable regions of anti-human IL-5 monoclonalantibody JES1-39D10, the production of which is described below, aredefined in the Sequence Listing by SEQ ID NOs: 1 and 2, respectively.The amino acid sequences predicted from these nucleotide sequences arealso defined in SEQ ID NOs: 1 and 2.

In the nucleotide sequence defined by SEQ ID NO: 1 (heavy chainsequence), bases I to 26 were derived from a polymerase chain reaction(PCR) primer. Therefore, the cloned sequence begins at base 27. Thecorresponding amino acid sequence from SEQ ID NO: 1 is that of themature polypeptide of V_(H), not including the leader or the firstfourteen residues of framework 1. In the nucleotide sequence defined bySEQ ID NO: 2 (light chain sequence), bases 1 and 2 were derived from aPCR primer. Therefore, the cloned sequence begins at base 3. Thecorresponding amino acid sequence is that of the leader and the maturepolypeptide of V_(L).

The CDRs of the heavy chain variable region of monoclonal antibodyJES1-39D10 as determined by the method of Kabat et al., supra, compriseamino acid residues 26-30, 45-60 and 93-100 of the amino acid sequencedefined by SEQ ID NO: 1. As determined by computer analysis of bindingsite loop structures as described below, the CDR structural loops of theheavy chain variable region of monoclonal antibody JES1-39D10 compriseamino acid residues 21-27, 47-50 and 93-101 of the amino acid sequencedefined by SEQ ID NO: 1.

Nucleotide sequences encoding the foregoing heavy chain CDRs comprisebases 76-90, 133-180 and 277-300 (Kabat determination) and bases 61-81,139-150 and 277-303 (loop analysis) of the nucleotide sequence definedby SEQ ID NO: 1.

The CDRs of the light chain variable region of monoclonal antibodyJES1-39D10 as determined by the method of Kabat et al., supra, compriseamino acid residues 44-54, 70-76 and 109-117 of the amino acid sequencedefined by SEQ ID NO: 2. As determined by computer analysis of bindingsite loop structures as described below, the CDR structural loops of thelight chain variable region of monoclonal antibody JES1-39D10 compriseamino acid residues 46-51, 70-72 and 111-116 of the amino acid sequencedefined by SEQ ID NO: 2.

Nucleotide sequences encoding the foregoing light chain CDRs comprisebases 130-162, 208-228 and 325-351 (Kabat determination) and bases136-222, 208-216 and 331-348 (loop analysis) of the nucleotide sequencedefined by SEQ ID NO: 2.

From the foregoing, it can be seen that the CDRs thus determined areencoded by from 9 to 48 bases. Useful DNAs for protein engineeringtherefore comprise from about 12 to 333 bases and from about 9 to 384bases of the nucleotide sequences defined by SEQ ID NOs: 1 and 2,respectively. Also of importance is the constant region for selection ofisotype for protein engineering.

If the CDRs of the invention are used to produce humanized antibodies bygrafting onto a human antibody, it may be desirable to include one ormore amino acid residues which, while outside the CDRs, are likely tointeract with the CDRs or IL-5 (Queen et al., supra).

The CDRs of the invention can also form the basis for the design ofnon-peptide mimetic compounds which mimic the functional properties ofantibody JES1-39D10. Methods for producing such mimetic compounds havebeen described by Saragovi et al. [Science 253:792 (1991)].

In addition to providing a basis for antibody humanization, theinformation in SEQ ID NOs: 1 and 2 can be used to produce single-chainIL-5 binding proteins comprising linked CDRs from the light and/or heavychain variable regions, as described by Bird et al. [Science 242:423(1988)], or biosynthetic antibody binding sites (BABS), as described byHuston et al. [Proc. Natl. Acad. Sci. USA 85:5879 (1988)]. Single-domainantibodies comprising isolated heavy-chain variable domains [Ward etal., Nature 341:544 (1989)] can also be prepared using the informationin SEQ ID NO: 1.

Two or more CDRs of the invention can also be coupled together in apolypeptide, either directly or by a linker sequence. One or more of theCDRs can also be engineered into another (non-immunoglobulin)polypeptide or protein, thereby conferring IL-5 binding capability onthe polypeptide or protein.

Polypeptides “comprising a heavy or light chain variable region of amonoclonal antibody having a sequence defined by SEQ ID NOs: 1 or 2, ora subsequence thereof”, are defined herein to include all of theforegoing CDR-containing embodiments.

DNAs which encode the heavy and light chain variable regions of antibodyJES1-39D10 or the CDRs therefrom can be prepared by. standard methodsusing the nucleic acid sequence information provided in SEQ ID NOs: 1and 2. For example, such DNA can be chemically synthesized using, e.g.,the phosphoramidite solid support method of Matteucci et al. [J. Am.Chem. Soc. 103:3185 (1981)], the method of Yoo et al. [J. Biol. Chem.764:17078 (1989)], or other well known methods.

Alternatively, since the sequence of the gene and the site specificitiesof the many available restriction endonucleases are known, one skilledin the art can readily identify and isolate the gene from the genomicDNA of the hybridoma producing monoclonal antibody JES1-39D10 and cleavethe DNA to obtain the desired sequences. The PCR method [Saiki et al.,Science 239:487 (1988)], as exemplified by Daugherty et al. [NucleicAcids Res. 19:2471 (1991)] can also be used to obtain the same result.Primers used for PCR can if desired be designed to introduce appropriatenew restriction sites, to facilitate incorporation into a given vector.

Still another method for obtaining DNAs encoding the heavy and lightchain variable regions of antibody JES1-39D10 entails the preparation ofcDNA, using mRNA isolated from the hybridoma producing monoclonalantibody JES1-39D10 as a template, and the cloning of the variableregions therefrom using standard methods [see, e.g., Wall et al.,Nucleic Acids Res. 5:3113 (1978); Zalsut et al., Nucleic Acids Res.8:3591 (1980); Cabilly et al., Proc. Natl. Acad. Sci. USA 81:3273(1984); Boss et al., Nucleic Acids Res. 12:3791 (1984); Amster et al.,Nucleic Acids Res. 8:2055 (1980); Moore et al., U.S. Pat. No.4,642,234].

Of course, due to the degeneracy of the genetic code, many differentnucleotide sequences can encode CDRs, polypeptides and antibodies havingamino acid sequences defined by SEQ ID NOs: 1 and 2 and the CDRstherein.

Similarly, humanized antibodies having amino acid sequences describedbelow can be encoded by many different DNAs.

The particular codons used can be selected for both convenientconstruction and optimal expression in prokaryotic or eukaryoticsystems. Such functional equivalents are also a part of this invention.Furthermore, those skilled in the art are aware that there can beconservatively modified variants of polypeptides and proteins in whichthere are minor amino acid substitutions, additions or deletions that donot substantially alter biological function [Anfinsen, Science 181:223(1973); Grantham, Science 185:862 (1974)].

Such conservatively modified variants of the amino acid sequencesdefined by SEQ ID NOs: 1 and 2 and of the humanized antibodies describedbelow are also contemplated by this invention. It is well within theskill of the art, e.g., by chemical synthesis or by the use of modifiedPCR primers or site-directed mutagenesis, to modify the DNAs of thisinvention to make such variants if desired.

It may also be advantageous to make more substantial modifications. Forexample, Roberts et al. [Nature 328:731 (1987)] have produced anantibody with enhanced affinity and specificity by removing two chargedresidues at the periphery of the combining site by site-directedmutagenesis.

Insertion of the DNAs encoding the heavy and light chain variableregions of antibody JES1-39D10 into a vector is easily accomplished whenthe termini of both the DNAs and the vector comprise compatiblerestriction sites. If this cannot be done, it may be necessary to modifythe termini of the DNAs and/or vector by digesting back single-strandedDNA overhangs generated by restriction endonuclease cleavage to produceblunt ends, or to achieve the same result by filling in thesingle-stranded termini with an appropriate DNA polymerase.Alternatively, any site desired may be produced by ligating nucleotidesequences (linkers) onto the termini. Such linkers may comprise specificoligonucleotide sequences that define desired restriction sites. Thecleaved vector and the DNA fragments may also be modified if required byhomopolymeric tailing or PCR.

Pharmaceutical compositions can be prepared using the antibodies,binding compositions or single-chain binding proteins of the invention,or anti-idiotypic antibodies prepared against such monoclonalantibodies, to treat IL-5-related diseases. Fragments of the antibodiessuch as Fab or Fv fragments, isolated heavy or light chains or fragmentstherefrom, and short polypeptides comprising, e.g., individual CDRregions, can also be used in such compositions.

Some of the compositions have IL-5 blocking or antagonistic effects andcan be used to suppress IL-5 activity. Such compositions comprise theantibodies, binding compositions or single-chain binding proteins of theinvention and a physiologically acceptable carrier.

Other compositions comprise anti-idiotypic antibodies prepared using themonoclonal antibodies of the invention as an antigen and aphysiologically acceptable carrier. These anti-idiotypic antibodies,which can be either monoclonal or polyclonal and are made by standardmethods, may mimic the binding activity of IL-5 itself. Thus, they maypotentially be useful as IL-5 agonists or antagonists.

Useful pharmaceutical carriers can be any compatible, non-toxicsubstance suitable for delivering the compositions of the invention to apatient. Sterile water, alcohol, fats, waxes, and inert solids may beincluded in a carrier. Pharmaceutically acceptable adjuvants (bufferingagents, dispersing agents) may also be incorporated into thepharmaceutical composition. Generally, compositions useful forparenteral administration of such drugs are well known; e.g. Remington'sPharmaceutical Science, 15th Ed. (Mack Publishing Company, Easton, Pa.,1980). Alternatively, compositions of the invention may be introducedinto a patient's body by implantable drug delivery systems [Urquhart etal., Ann. Rev. Pharmacol. Toxicol. 24:199 (1984)].

EXAMPLE

In the following non-limiting Example used for the purpose ofillustration, percentages for solids in solid mixtures, liquids inliquids, and solids in liquids are given on a wt/wt, vol/vol and wt/volbasis, respectively, unless otherwise indicated. Sterile conditions weremaintained during cell culture.

General Methods and Reagents

Unless otherwise noted, standard recombinant DNA methods were carriedout essentially as described by Maniatis et al., Molecular Cloning: ALaboratory Manual, 1982, Cold Spring Harbor Laboratory.

Small scale isolation of plasmid DNA from saturated overnight cultureswas carried out according to the procedure of Birnboim et al. [NucleicAcids Res. 7:1513 (1979)]. This procedure allows the isolation of asmall quantity of DNA from a bacterial culture for analytical purposes.Unless otherwise indicated, larger quantities of plasmid DNA wereprepared as described by Clewell et al. [J. Bacteriol. 110:1135 (1972)].

Specific restriction enzyme fragments derived by the cleavage of plasmidDNA were isolated by preparative electrophoresis in agarose. Gelsmeasuring 9×5 ½ cm were run at 50 mA for 1 hour in Tris-Borate buffer(Maniatis et al., supra, p. 454) and then stained with 0.5 μg/mlethidium bromide to visualize the DNA. Appropriate gel sections wereexcised, and the DNA was electroeluted (Maniatis et al., supra, p. 164).After electroelution, the DNA was phenol extracted (Maniatis et al.,supra, p. 458) and ethanol precipitated at −20° C. (Maniatis et al.,supra, p. 461).

Restriction enzymes and T4 DNA ligase were purchased from New EnglandBiolabs (Beverly, Mass.). Superscript RNAse H- reverse transcriptase wasfrom BRL/GIBCO (Gaithersburg, Md.), Taq DNA polymerase from Stratagene(LaJolla, Calif.), DNA polymerase Klenow fragment from Pharmacia LKBBiotechnology, Inc. (Piscataway, N.J.), calf intestinal phosphatase fromBoehringer Mannheim Biochemicals (Indianapolis, Ind.) and RNAsin fromPromega (Madison, Wis.). All enzymes were used in accordance with themanufacturers' instructions. The Sequenase version 2.0 sequencing systemwas obtained from United States Biochemical (Cleveland, Ohio).

Deoxynucleotide triphosphates and oligo dT₁₂₋₁₈ primer were fromPharmacia LKB Biotechnology, bovine serum albumin (BSA) from BoehringerMannheim Biochemicals and re-distilled phenol from BRL/GIBCO.

Plasmid vectors pSV.Sport and pUC19, and competent E. coli strainDH5-alpha (Max Efficiency) were from BRL/GIBCO. COS-7 cells (ATCC CRL1651) were obtained from the American Type Culture Collection, Bethesda,Md. Tissue culture media, fetal bovine serum (FBS) and supplements werefrom BRL/GIBCO.

Recombinant human IL-5 was expressed in Chinese hamster ovary (CHO)cells by standard methods using plasmid pDSRG (ATCC 68233) and purifiedby immunoaffinity chromatography.

Rabbit antiserum against recombinant human IL-5 was made by standardmethods. Biotinylated goat anti-rabbit IgG was obtained from VectorLabs, Burlingame, Calif., while streptavidin-alkaline phosphataseconjugate was a product of BRL/GIBCO. Biotinylated rabbit anti-rat IgGwas from Jackson Labs, West Grove, Pa. Nitro blue tetrazolium (NBT) and5-bromo-4-chloro-3-indolyl phosphate (BCIP) were obtained fromBRL/GIBCO.

A highly sensitive ELISA AMPLIFICATION SYSTEM® used as the substrate formicrotiter plate ELISA (enzyme-linked immunosorbent assay)determinations was obtained from BRL/GIBCO.

Cell Culture

The hybridoma cell line producing monoclonal antibody JES 1 -39D10 wasproduced as described by Denburg et al. [Blood 77:1462 (1991)] andinitially maintained in Dulbecco's Modified Eagle's medium (DMEM/highglucose; GIBCO, Bethesda, Md.) supplemented with 5% FBS, 2 mM glutamineand 10 units/ml penicillin/streptomycin in a humidified 37° C. chamberwith 5% CO₂. Subsequently, the cell line was adapted to serum-freeculture by replacing the serum with 1×HB101 supplement (Hana Biologics,Alameda, Calif.).

Characterization of Monoclonal Antibody JES1-39D10 Purification

Medium conditioned by the cell line producing antibody 39D10.11 (asubclone of JES1-39D10) was harvested, filtered and applied to anantigen affinity column prepared by coupling recombinant human IL-5 toAFFIGEL-15® resin (BioRad, Richmond, Calif.). The column was then washedsequentially with phosphate buffered saline (PBS) and PBS with 0.5 MNaCl, and then equilibrated with PBS. Antibody 39D10.11 was eluted fromthe column with 0.2 M glycine buffer, pH 2.95, after which the elutedprotein was neutralized immediately with 1 M Tris-HCl, pH 8.

The purified protein was subjected to polyacrylamide gel electrophoresis(SDS-PAGE) in a 15% gel under reducing conditions essentially asdescribed by Laemmli [Nature 227:680 (1970)], to separate the heavy andlight chains. Both chains were recovered by transfer onto an IMMOBILON®membrane (a PVDF membrane from Millipore, Bedford, Mass.), essentiallyusing the electroblotting method of Matsudaira [J. Biol. Chem. 261:10035(1987)].

Bands corresponding to the heavy and light chains were excised from themembrane following staining with Coomassie Brilliant Blue and processedfor N-terminal sequencing using an Applied Biosystems Model 477Aprotein-peptide sequencer. Sequencing of the isolated heavy and lightchains blotted onto the IMMOBILON® membrane was carried out essentiallyas described by Yuen et al. [Biotechniques 7:74 (1989)].

The heavy chain could not be sequenced under standard conditions. Thelight chain was sequenced up to 15 cycles. Comparison of the sequenceobtained with published data confirmed the identity of a ratimmunoglobulin light chain.

Antibody Isotyping

Isotyping of antibody JES1-39D10 carried out using a kit from Zymed (SanFrancisco, Calif.) revealed that heavy chain was a gamma 2a isotype andthe light chain was a kappa isotype (γ2a/κ isotype).

Effect on Biological Activity

Monoclonal antibody JES1-39D10 strongly inhibits the biological activityof recombinant human IL-5 (Denburg et al., supra). Studies have shownthat the antibody produces this effect by inhibiting the binding of theIL-5 to its cellular receptors.

PCR Cloning

Oligonucleotide Primer Design and Cloning Strategy

Briefly, oligonucleotide primers were prepared corresponding to theknown amino- and carboxyl-termini of rat IgG2a and kappa heavy and lightchain sequences [Reichmann et al., Nature 332:323 (1988); Bruggemann etal., Proc. Natl. Acad. Sci. USA 83:6075 (1986); Hellman et al., Gene40:107 (1985)]. Using these primers, cDNA fragments of the completelight chain and a truncated heavy chain were isolated using the PCRmethod [Saiki et al., Science 239:487 (1988)]. The cDNA fragments weresequenced and confirmed by comparison with other clones generated usingPCR primers designed from different regions of the cDNAs. The deducedamino acid sequence of the light chain was verified by the sequencing ofthe first 15 N-terminal amino acid residues of the JES1-39D10 lightchain.

Oligonucleotide Synthesis

Based upon the IgG2a/kappa isotype of antibody JES 1-39D10,oligonucleotide primers having sequences defined in the Sequence Listingwere synthesized by standard methods using an Applied BioSystems Model380A or 380B Synthesizer.

The designations of these primers (with the first letter correspondingto the synthesizer model used) and the corresponding sequenceidentification numbers are as follows:

Oligonucleotide SEQ ID NO. B2051CC 3 B2031CC 4 A2064CC 5 A2065CC 6B2137CC 7 B2108CC 8 B2101CC 9 B1852CC 10 

For the heavy chain of antibody JES1-39D10, the 5′ end primer B2051CCwas derived from a segment of a rat IgG2a sequence published byReichmann et al., supra, encompassing the sixth through the fourteenthamino acid residues of the mature heavy chain polypeptide. Tworestriction sites, XbaI and HindII, were added at the 5′ end of theprimer to facilitate cloning. The 3′ end primer B2031CC was based upon asegment of the published rat IgG2a constant region three sequence(Bruggemann et al., supra). Three restriction sites, SmaI, EcoRI andSalI, were added for cloning purposes.

For the light chain, both the 5′ and 3′ end primer sequences werederived from a published rat kappa mRNA sequence (Hellman et al.,supra). Primer A2064CC encompasses a partial 5′ untranslated region andthe first two nucleotides of the translation initiation codon. A HindIIIand a BamHI site were added to facilitate cloning. The 3′ end primerA2065CC was based upon a segment of a published rat kappa immunoglobulinlight chain 3′ untranslated region (3′ UTR). This segment encompassesthe last two nucleotides of the stop codon and 22 3′ UTR nucleotidesimmediately 3′ of the stop codon. EcoRI and PstI sites were added tofacilitate cloning.

PCR Reaction Conditions

Total cytoplasmic RNA was isolated from the hybridoma cell lineproducing antibody JES1-39D10, essentially as described by Cathala etal. [DNA 2:329 (1983)]. First-strand cDNA synthesis was carried outusing the isolated RNA as the template, essentially as described by byLarrick et al. [Bio/Technology 7:934 (1989)]. The cDNA product was thensubjected to PCR in a 50 μl volume reaction mixture with a 50 μlparaffin oil overlay, in a 0.5 ml Eppendorf tube.

The reaction mixture for heavy chain synthesis contained 26.5 μl of H20,5 μl of Taq (Thermus aquaticus) DNA polymerase buffer [finalconcentrations in the reaction: 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5mM MgCl2, 0.01% (w/v) gelatin], 5 μl of 1.25 mM dNTP, 4 μl of primerB2051CC (60 pmol), 4 μl of primer B2031CC (60 pmol), 5 μl of cDNA(containing half of the first-strand cDNA derived from 3-6 μg total RNA)and 0.5 μl of Taq polymerase (2.5 units). The reaction mixture for lightchain synthesis was similar, except that the primers used were A2064CCand A2065CC.

The reaction was carried out in a PHC-1 Thermocycler (Techne, Princeton,N.J.) with 30 cycles of: 94° C., 1.5 minutes for denaturation; 50° C., 2minutes for annealing; and 72° C., 3.5 minutes for synthesis. At the endof the 30th cycle, the reaction mixture was incubated another 9 minutesat 72° C. for extension.

The PCR mixture was subjected to electrophoresis in a 1%agarose/Tris-Borate gel containing 0.5 μg/ml ethidium bromide. DNAfragments having the expected sizes (approximately 1.5 kilobases for theheavy chain, approximately 0.7 kilobases for the light) were excisedfrom the gel and purified by electroelution.

Verification of cDNA Clones

DNA Sequencing

Following recovery from the gel, ethanol precipitation and restrictionendonuclease cleavage, the PCR-generated putative heavy and light chaincDNAs were cloned into an appropriate vector for sequencing.

The putative heavy and light chain cDNAs were cloned separately into thepUC19 vector as HindIII/EcoRI and BamHI/PstI fragments, respectively.The ligated plasmids were then transformed into E. coli strain DH5-alphausing standard methods. Transformant colonies were isolated, and plasmidDNA from at least two clones each for the heavy and light chains wasprepared from the transformants for sequencing.

Comparison of the variable region sequences thereby obtained (definedfor the heavy and light chains by SEQ ID NOs: 1 and 2, respectively)with the published immunoglobulin sequences revealed a high degree ofhomology in the framework and in the positioning of the putative CDRs asdetermined by the method of Kabat et al., supra. Moreover, theN-terminal amino acid sequence predicted from the light chain cDNAsequence agreed with the experimentally-determined N-terminal sequenceof the light chain of antibody JES1-39D10.

Expression Plasmid Construction

To confirm that the isolated cDNAs encoded proteins that couldspecifically bind to human IL-5, cDNA encoding a full-length putativeheavy chain of antibody JES1-39D10 was reconstructed by PCR. PrimersB2137CC (SEQ ID NO: 7) and B2108CC (SEQ ID NO: 8) were designed tomodify the initially-cloned truncated heavy chain cDNA. These primerssupplied sequences encoding the leader peptide, five missing amino acidresidues at the N-terminus of the variable region, and missing residuesat the C-terminus of the constant region.

A vertebrate consensus translational initiator sequence [Kozak, NucleicAcids Res. 20:8125 (1987)] was added to the 5′ end of the coding cDNA tofacilitate translation. To facilitate cloning, SalI and EcoRIrestriction sites were introduced into the 5′ and 3′ ends, respectively,of the resulting cDNA.

The putative light chain cDNA was also re-engineered, using primersB2101CC (SEQ ID NO: 9) and B1852CC (SEQ ID NO: 10). This was doneprimarily to modify the restriction sites at the ends for expressionplasmid construction. The vertebrate consensus translational initiatorsequence was also introduced into this cDNA.

PCR was carried out and the DNA products were isolated as describedabove, and the DNAs were cleaved with SalI and EcoRI. The heavy andlight chain cDNAs were then ligated separately into expression vectorpSRS (ATCC 68234) which had been similarly cleaved. Plasmid pSRScontains an SRa promoter and an SV40 origin of replication to enablereplication and expression of the cDNA inserts in COS cells.

Transfection

Prior to transfection, COS cells were propagated in DMEM/high glucosesupplemented with 10% FBS and 6 mM glutamine. Exponentially grown cellswere trypsinized, washed with fresh medium, and then resuspended infresh medium at a density of 2×107 cells/ml.

Electroporation was carried out using a GENEPULSER® (BioRad, Richmond,Calif.) according to the manufacturer's instructions. Briefly, a 250 μlaliquot of the COS cell suspension was dispensed into each cuvette.About 10 μg of CIRCLEPREP® (Bio101, LaJolla, Calif.)-purified plasmidDNA was added in a volume of less than 50 μl to the cuvettes and wasmixed with the cells. DNA samples used included pSRS with the heavychain cDNA insert (pSRS-H), pSRS with the light chain cDNA insert(pSRS-L), an equal amount mixture of pSRS-H and pSRS-L, and unmodifiedpSRS.

A 0.2 volt electrical pulse was delivered at 960 μF with a capacityextender. The cells were then plated in 60 mm culture dishes, fed with 5ml of fresh medium and incubated at 37° C. in a 5% CO₂ incubator. After16 hours, the medium was removed by aspiration and replaced withserum-free medium. Incubation was continued for an additional 72 hours,after which the medium was harvested.

Immunoblot Analysis

The serum-free media conditioned by the transfected cells wereconcentrated about ten fold by centrifugation in CENTRICON® tubes(Amicon, Danvers, Mass.), after which they were subjected toelectrophoresis in a 15% precast SDS polyacrylamide gel (IntegratedSeparation Systems, Hyde Park, Mass.) under non-reducing conditions. Twoidentical sets of samples were applied to the same gel. Two identicalmolecular weight marker protein mixtures (containing 97.4, 68, 43, 29,18.4 and 14.3 kilodalton proteins) were also run in the gel.

Following electrophoresis, the separated bands were transferred onto anitrocellulose membrane using a Semi-dry Electroblotter (IntegratedSeparation Systems, Hyde Park, Mass.). The membrane was then incubatedat room temperature with 3% BSA in PBS, after which the membrane was cutinto two pieces. Each piece contained a complete set of the samples.

One piece of the membrane was used in a first analysis to detect rat IgGchain expression in the conditioned media. This was accomplished bytreating the membrane in turn at room temperature: once with a 1:200dilution of biotinylated rabbit anti-rat IgG in TBST buffer [10 mMTris-HCl, pH 7.4, 150 mM NaCl and 0.05% TWEEN-20®(polyoxyethylenesorbitan monolaurate)] with 3% BSA for 45 minutes, threetimes with TBST buffer alone for 15 minutes, once with a 1:10,000dilution of streptavidin-alkaline phosphatase conjugate in TBST bufferfor 30 minutes, three times with TBST buffer alone for 15 minutes, andonce with alkaline phosphatase substrate (44 μl of NBT and 33 μl of BCIPin 10 ml of an alkaline phosphatase buffer containing 100 mM Tris-HCl,pH 9, 100 mM NaCl, 5 mM MgCl₂) for 10-30 minutes. The membrane piece wasthen examined for color development after rinsing with distilled water.

The other piece of the membrane was used in a second analysis todetermine whether a human IL-5 binding protein was present in any of theconditioned media. This piece was treated sequentially at roomtemperature: once with 1 μg/ml recombinant human IL-5 in TBST buffer for1 hour, three times with TBST buffer alone for 15 minutes, once with a1:3,000 dilution of a rabbit antiserum against recombinant human IL-5 inTBST buffer for 1 hour, three times with TBST buffer alone for 15minutes, once with a 1:200 dilution of biotinylated goat anti-rabbit IgGin TBST buffer for 1 hour, three times with TBST buffer alone for 15minutes, once with a 1:10,000 dilution of streptavidin-alkalinephosphatase conjugate in TBST buffer for 30 minutes, three times withTBST buffer alone for 15 minutes, and once with the alkaline phosphatasesubstrate for 30 minutes. The membrane piece was then examined for colordevelopment.

The results of the first analysis showed that only the mediumconditioned by the transformant cotransfected with both plasmids pSRS-Hand pSRS-L produced detectable bands. The pattern of bands was asexpected for immunoglobulins. Staining was not observed in the lanescontaining conditioned media from cells transfected with heavy or lightchain alone, indicating either low quantities of protein or an inabilityof the antibody to recognize individual heavy and light chains. Heavychain was not expected to be present in the conditioned media, since itis usually not secreted in the absence of a light chain [Bole et al., J.Cell Biol. 102:1558 (1986)].

In the second analysis, strong IL-5 binding activity was observed in thesample from the medium conditioned by the transformant cotransfectedwith both plasmids pSRS-H and pSRS-L. This was noted as a major stainedband in a nonreducing SDS gel migrating with an apparent molecularweight of about 160 kilodaltons, a size expected for a complete antibodymolecule.

Enzyme-Linked Immunosorbent Assay

To confirm the ability of the recombinant monoclonal antibody tospecifically bind to human IL-5, a 96-well immunoassay (EIA) plate wascoated with 50 μl aliquots of 3% BSA alone, or recombinant human IL-5 (5μg/ml, followed by blocking with the BSA solution). All coating was donefor approximately 2 hours at 4° C. Aliquots (50 μl) of media conditionedby the various COS cell transformants described above or by thehybridoma cell line producing antibody JES1-39D10 were then added to thewells, and the plate was incubated for 2 hours at 4° C.

Following the incubation, the media in the wells were replacedsequentially using 300 μl aliquots at room temperature: three times withTBST buffer alone for 15 minutes, once with biotinylated rabbit anti-ratIgG for approximately 2 hours, three times with TBST alone for 15minutes, once with streptavidin-alkaline phosphatase conjugate in TBSTbuffer for 30 minutes, three times with TBST buffer alone for 15minutes, and then developed with the BRL ELISA AMPLIFICATION SYSTEM® for5-15 minutes, all reagent dilutions being as described above.

The results of this assay showed that both the natural JES1-39D10antibody (from the medium conditioned by the hybridoma) and therecombinant antibody (from the medium conditioned by the transformantcotransfected with both plasmids pSRS-H and pSRS-L) were retained inwells coated with IL-5, as evidenced by a strong color development. Nosignificant color development above that of background controls(containing no antibody protein) was observed when IL-5 was absent, orwhen the test samples were conditioned media of cells transfected withvectors encoding heavy or light chains alone.

Antibody Humanization

Homology Modeling

Using the methods described above, it was determined that antibodies HILand LAY were optimal human framework candidates. HIL or LAY heavy andlight chain pairs and combinations thereof were first pursued.

A listing of potential minimal and maximal JES 1 -39D10 residues thatcould be grafted into the framework sequences were determined by theabove-described methods to be as shown in the following Table.

Residues^(a) VH Minimal List: 21-27, 47-50, 66, 93-101 VH MaximalList^(b): 19, 28, 29, 30, 32, 40, 45, 46, 51, 52-60, 71, 89, 92 VLMinimal List: 46-51, 68, 70-72, 84, 91, 109, 110-116 VL MaximalList^(b): 44, 45, 52-54, 56, 66, 73-76, 88, 89, 117 ^(a)Residues for VHand VL refer to the residue numbers in SEQ ID NO: 1 and SEQ ID NO: 2,respectively. ^(b)The VH and VL Maximal Lists include the correspondingMinimal Lists and the further indicated residues.

Specific constructs described below contained the following residuesfrom the foregoing Table:

Humanized Antibody Residues CMX5-1 VH HIL Maximal; VL LAY Maximal CMX5-2VH HIL Maximal; VL LAY Maximal (less structural CDR loop H-1 N-terminus)CMX5-3 VH LAY Maximal; VL LAY Maximal CMX5-4 VH HIL Minimal; VL LAYMinimal CMX5-5 VH HIL Kabat; VL LAY Kabat CDRs only CDRs only

Since the intended use of the humanized antibodies was theneutralization of the biological activity of soluble human IL-5, γ4 waschosen as the constant region for the humanized antibodies. This isbecause γ4 is the least potent of the four human immunoglobulin isotypesin triggering complement fixation. The human counterpart of the rat Kisotype was chosen for the constant region of the humanized lightchains.

Construction of Humanized Antibody CMX5-1 Synthetic CMX5-1 DNA wasconstructed using a combination of PCR and conventional cloningtechniques. The V region was divided into three segments, each of whichwas designed to contain designated restriction sites at the 5′ and 3′ends. Codons were chosen which occur with relatively high frequency inmammalian genes. Palindromic and repeat sequences were avoided orreplaced by silent changes in the design of oligonucleotides, tominimize formation of unanticipated secondary structures that may causesequence rearrangements or deletions.

Oligonucleotides corresponding to the entire heavy chain variable region(V_(H)) of CMX5-1 were synthesized by standard methods.

The designations of these oligonucleotides and the corresponding SEQ IDNOs defining their sequences are as follows:

Oligonucleotide SEQ ID NO. B2474CC 11 B2419CC 12 B2420CC 13 B2475CC 14B2477CC 15 B2479CC 16

Pairs of oligonucleotides B2474CC and B2419CC, B2420CC and B2475CC,B2477CC and B2479CC were heat-denatured, annealed, and incubated withTaq polymerase or Pfu (Stratagene, La Jolla, Calif.). In the polymerasechain reactions (PCRs), the two oligonucleotides in each pair werecomplementary to each other by about 24 to 30 nucleotides. Therefore,each oligonucleotide served as the template for the other.

The PCRs were carried out for 18 cycles, after which the three resultingDNA fragments, corresponding to the three consecutive segments of V_(H),designated V_(H)1, V_(H)2 and V_(H)3, were electrophoresed in an agarosegel and purified by electroelution.

The relative order of the three V_(H) DNA fragments, restriction sitesfor cloning, and the multicloning-site map of cloning vector pSV.Sportwere as follows:

Fragment Restriction Sites PCR Primers VH1 EcoRI  SpeI B2474CC + B2419CCVH2 SpeI  XbaI B2420CC + B2475CC VH3 EcoRI/XbaI  SalI/ApaI/SstIB2477CC + B2479CC

Multi-cloning Sites of pSV.Sport

PstI/KpnI/RsrII/EcoRI/SmaI/SalI/I/SstI/SpeI/NotI/XbaI/BamHI/HindIII/SnaBI/MluI

Fragment V_(H)1 was restricted with enzymes EcoRI and SpeI and clonedinto vector pSV.Sport. Fragment V_(H)2 was subsequently joined to V_(H)1in pSV.Sport by directional insertion at SpeI and XbaI sites. FragmentV_(H)3 was separately cloned into pSV.Sport as anEcoRI/XbaI-SalI/ApaI/SstI fragment. The three fragments were verified byDNA sequencing.

Full-length CMX5-1 V_(H) cDNA was assembled by first joining V_(H)3 to agenomic DNA of the γ4 H-chain constant region (CH) and then attachingthe V_(H)3-C_(H) fragment to the V_(H)1-V_(H)2 fragment, as is describedmore fully below.

CMX5-1 V_(L) CDNA was assembled in a similar manner, using six syntheticoligonucleotide primers. These oligonucleotides and the correspondingSEQ ID NOs defining their sequences are as follows:

Oligonucleotide SEQ ID NO. B2425RCC 17 B2426CC 18 B2427CC 19 B2458CC 20B2459CC 21 B2460CC 22

Three fragments, designated V_(L)1, V_(L)2, and V_(L)3, were derivedfrom oligonucleotide pairs B2425RCC and B2426CC, B2427CC and B2458CC,B2459CC and B2460CC, respectively. These DNA fragments, which correspondto the three consecutive segments of V_(L), were purified by agarose gelelectrophoresis and electroelution.

The relative order of the three V_(L) DNA fragments, restriction sitesfor cloning, and the multicloning-site map of cloning vector pUC19 wereas follows:

Fragment Restriction Sites PCR Primers VL1 PstI  BstEII/XbaI B2425RCC +B2426CC VL2 PstI/BstEII  BamHI B2427CC + B2458CC VL3 BamHI  SalIB2459CC + B2460CC

Multi-cloning sites of pUC19

EcoRI/SacI/KpnI/SmaI/BamHI/XbaI/SalI/PstI/SphI/HindIII

V_(L)1, V_(L)2 and V_(L)3 were first cloned separately into vector pUC19as PstI-BstEII/XbaI, PstI/BstEII-BamHI and BamHI-SalI fragments,respectively. The identities of the three fragments were verified by DNAsequencing. V_(L)1 and V_(L)2 were then assembled together in pUC19 byinserting V_(L)2 as a BstEII-BamHI fragment into the vector alreadycontaining V_(L)1.

The assembly of full-length V_(L) cDNA was accomplished by first joiningV_(L)3 to light-chain constant region (C_(L)) and then joining theV_(L)1-V_(L)2 and V_(L)3-C_(L) fragments, as is described more fullybelow.

To facilitate synthesis and secretion of the humanized antibodies, aleader peptide was attached to the amino-termini of both the mature H-and L-chain polypeptides. The amino acid and nucleotide sequences ofthis leader are those of the leader of the anti-CAMPATH-1 antibodies(Reichmann et al., supra). The coding sequence for the leader peptide(Reichmann et al., supra) was incorporated into both fragments V_(H)1and V_(L)1.

In an effort to construct DNA encoding a full-length antibody H-chain,the V_(H) synthetic cDNA was combined with human γ4 constant-regiongenomic DNA (ATCC 57413) using ApaI restriction cleavage and ligation.This procedure was initiated by digesting plasmid pSV.Sport containingV_(H)3 with NotI followed by treatment with Klenow DNA polymerase(Boehringer Mannheim) to generate blunt ends. The resulting DNA wasethanol-precipitated, resuspended, and digested with ApaI. Thisrestricted plasmid DNA was ligated with the ApaI/SmaI restrictionfragment of the genomic γ4 constant region.

The V_(H)3 -C_(H) genomic DNA was then excised as an XbaI/HindIIIfragment and inserted into pSV.Sport already containing V_(H)1-V_(H)2,thereby completing assembling of the full-length heavy chain DNA.

In an effort to produce the antibody chains, plasmids containing the H-and L-chain DNAs were co-transfected into COS cells. Secretedimmunoglobulin was undetectable, however, following analysis of theconditioned medium by ELISA or Western blotting. As an alternative, ahuman _(γ)4 constant-region cDNA was designed and constructed to replacethe genomic DNA.

Six oligonucleotide PCR-primers were synthesized for this purpose bystandard methods. The designations of these oligonucleotides and thecorresponding SEQ ID NOs defining their sequences are as follows:

Oligonucleotide SEQ. ID NO. B2491CC 23 B2498CC 24 B2499CC 25 B2597CC 26B2598CC 27 B2656CC 28

Primers B2491CC, B2499CC and B2598CC correspond to the plus strand of_(γ)4 constant region cDNA. Primers B2498CC, B2597CC and B2656CCcorrespond to the minus strand. Using human _(γ)4 genomic DNA as thetemplate, three consecutive double-stranded DNA fragments encompassingthe entire _(γ)4 constant-region coding cDNA were generated by PCR.

The three C_(H) DNA segments, restriction sites for cloning, and primersused were as follows:

Segment Restriction Sites PCR Primers CH A. SalI  EcoRI B2491CC +B2498CC CK B. EcoRI  XhoI/SalI B2499CC + B2500CC CH C. SalI/XhoI  NotIB2598CC + B2656CC

Segment A was cloned into pUC19 as a SalI-EcoRI restriction fragment.Segment C, as a SalI/XhoI-NotI restriction fragment, was cloned intopSV.Sport. Segment B, as an EcoRI-XhoI/SalI fragment, was cloned intopSV.Sport already containing segment C. All three segments were verifiedby DNA sequencing.

The _(γ)4 cDNA was assembled by excising segment A with PstI and EcoRI,and cloning this fragment into pSV.Sport already containing segments Band C. The restriction map of the human _(γ)4 C_(H) cDNA and itsrelative position in pSV.Sport multi-cloning sites are as follows:

          A       B      CPstI/SalI---EcoRI---XhoI---NotI/HindIII/SnaBI/MluI

The _(γ)4 C_(H) cDNA was excised as a SalI - - - HindIII fragment toreplace the genomic _(γ)4 fragment in the previously describedfull-length H-chain construct. The final product was a full-lengthH-chain coding cDNA, cloned in vector pSV.Sport.

For stable transfection, plasmid pSV.Sport containing full-length heavychain cDNA was digested with KpnI and then treated with T4 polymerase toblunt the ends, and then digested with SnaBI. The resulting DNA fragmentwas isolated by agarose gel electrophoresis followed by purificationwith a GENECLEAN® DNA purification kit (Bio 101, La Jolla, Calif.), andblunt-end ligated into SmaI-treated plasmid pSRS or pDSRG. The finalconstructs were designated pSRSMPA5H (FIG. 1) and pDSRGMPA5H (FIG. 2).

The amino terminus of the heavy (H) chain of antibody HIL had previouslybeen reported to be chemically blocked [Chiu et al., Biochemistry 18:554(1977)]. A glutamine codon was assumed as the amino-terminal residue andwas used for the H chains of recombinant antibodies CMX1, 2, 4, and 5.In the H chain of antibody CMX5-3, the N-terminal alanine of the LAY Hchain was replaced by a glutamic acid residue following comparisons ofthe LAY H chain amino-terminal residue with corresponding residues ofother human H chain sequences in subgroup III, of which the LAY H chainis a member.

To facilitate construction of full-length variant L-chain cDNAs, thecodon of glutamic acid residue 105 was replaced by an aspartic-acidcodon to create a SalI restriction site near the junction of V_(L) andC_(L). This modification enabled substitution of V_(L) variants ascassettes in the pSV.Sport-based CMX5-1 L-chain expression plasmid.

A human kappa light-chain cDNA was initially constructed based onsequence information from human antibody REI [Epp et al., Eur. J.Biochem. 45:513 (1974)]. Seven synthetic oligonucleotide primers wereprepared for this purpose, the designations and corresponding SEQ ID NOsof which were as follows:

Oligonucleotide SEQ ID NO. B2262CC 29 B2281CC 30 B2293CC 31 B2294CC 32A2495CC 33 A2496CC 34 B2704CC 35

Four of the oligonucleotide primers were synthesized to encompass afull-length kappa light chain cDNA. These oligonucleotides (B2262CC,B2281CC, B2293CC and B2294CC) were mixed and extended in a singlepolymerase chain reaction. After the PCR, the product was purified byagarose electrophoresis and electroelution, cloned into pUC19, andanalyzed by DNA sequencing. All sequenced clones containedmisincorporated bases.

PCR primers A2495CC and A2496CC were then synthesized to generate acorrect human κ L-chain constant region. The sequence of A2495CCencompassed the last four amino acid codons of the human LAY antibodyV_(L) framework, and the beginning of the human κ constant region.Primer A2496CC corresponded to the carboxyl terminus of the human κconstant region.

PCR was carried out using an aberrant full-length L-chain clone andprimers A2495CC and A2496CC, and the PCR product was cloned into vectorpSV.Sport. Sequencing analysis showed, however, that the construct againcontained misincorporated bases. This new error was corrected by anadditional PCR using oligonucleotide primers A2495CC and B2704CC. Theproduct thereby obtained was cloned as a SalI/HindIII restrictionfragment into pUC19 that already carried the V_(L)3 fragment. A correctκ light-chain constant-region cDNA was thus obtained.

To assemble a full-length L chain cDNA, V_(L)1-V_(L)2 was excised as ablunt-ended HindIII/BamHI fragment and inserted into SmaI/BamHI-cleavedpUC19 containing the V_(L)3-C_(L) fragment. The full-length light-chainfragment was excised as a PstI/EcoRI fragment from pUC19 and clonedseparately into expression vectors pDSRG and pSRS, to generate plasmidsdesignated pDSRGMPA5L (FIG. 3) and pSRSMPA5L (FIG. 4), respectively.

The amino acid sequences of the heavy and light chain variable regionsof antibody CMX5-1 are defined in the Sequence Listing by SEQ ID NO: 36and SEQ ID NO: 37, respectively. Because amino acid residues 1-19 ofthese sequences comprise a secretory leader that is cleaved duringpost-translational processing, the actual variable region sequences aredefined by the sequences of SEQ ID NO: 36 and SEQ ID NO: 37, beginningwith residue 20.

Construction of Humanized Antibody CMX5-2

To construct humanized antibody CMX5-2, complementary oligonucleotidesdesignated B3194CC and B3195CC (sequences defined by SEQ ID NO: 38 andSEQ ID NO: 39, respectively), were synthesized and annealed to form aBglII/SpeI fragment to replace the 42 bp BglII/SpeI fragment of CMX5-1heavy chain in pSV.Sport. The replaced region was verified by DNAsequencing.

The amino acid sequences of the heavy and light chain variable regionsof antibody CMX5-2 are defined in the sequence listing by SEQ ID NO: 40and SEQ ID NO: 41, respectively. Because amino acid residues 1-19 ofthese sequences comprise a secretory leader that is cleaved duringpost-translational processing, the actual variable region sequences aredefined by the sequences of SEQ ID NO: 40 and SEQ ID NO: 41, beginningwith residue 20.

Construction of Humanized Antibody CMX5-3

To construct CMX5-3 V_(H), four oligonucleotides were synthesized havingamino acid sequences based on antibody JES1-39D10 CDR and human LAYV_(H) framework sequences. The designations and corresponding SEQ ID NOsof these oligonucleotides were as follows:

Oligonucleotide SEQ ID NO. B2784CC 42 B2785CC 43 B2786CC 44 B2921CC 45

PCRs were performed using sets of oligonucleotides B2784CC and B2785CCand B2786CC and B2921CC, and the products were restricted to generate aPstI/SpeI fragment and an XbaI/SalI fragment. These DNA fragments wereused to replace the V_(H)1 and V_(H)3 fragments, respectively, of theantibody CMX5-1 H chain cDNA in pSV.Sport.

The amino acid sequences of the heavy and light chain variable regionsof antibody CMX5-3 are defined in the sequence listing by SEQ ID NO: 46and SEQ ID NO: 47, respectively. Because amino acid residues 1-19 ofthese sequences comprise a secretory leader that is cleaved duringpost-translational processing, the actual variable region sequences aredefined by the sequences of SEQ ID NO: 46 and SEQ ID NO: 47, beginningwith residue 20.

Construction of Humanized Antibody CMX5-4

Antibody CMX5-4 V_(H) was constructed in a manner analogous to that usedto construct antibody CMX5-1. Three sets of overlapping oligonucleotideswere synthesized for this purpose, the designations (and defining SEQ IDNOs) of which were as follows:

Oligonucleotide SEQ ID NO. B2924CC 48 B2925CC 49 B2926CC 50 B2927CC 51B2928CC 52 B2929CC 53

Using sets of oligonucleotides B2924CC and B2925CC, B2926CC and B2927CC,and B2928CC and B2929CC, three corresponding DNA fragments weresynthesized by PCR extension reactions. The three fragments were cloned,sequenced, and assembled in pSV.Sport by restriction digestions andligations.

CMX5-4 V_(L) was constructed by first annealing oligonucleotidesdesignated B3093XY and B3094XY (sequences defined by SEQ ID NO: 54 andSEQ ID NO: 55, respectively) and then extending the 3′ ends of eacholigonucleotide by PCR. The product was gel-purified, BstEII and SalIrestricted, and used to replace the BstEII/SalI fragment in antibodyCMX5-1 L chain cDNA.

The amino acid sequences of the heavy and light chain variable regionsof antibody CMX5-4 are defined in the sequence listing by SEQ ID NO: 56and SEQ ID NO: 57, respectively. Because amino acid residues 1-19 ofthese sequences comprise a secretory leader that is cleaved duringpost-translational processing, the actual variable region sequences aredefined by the sequences of SEQ ID NO: 56 and SEQ ID NO: 57, beginningwith residue 20.

Construction of Humanized Antibody CMX5-5

CMX5-5 V_(H) DNA was constructed by synthesizing two DNA fragments usingpairs of oligonucleotides designated B3136CC and B3137CC, and B3138CCand B3202CC, the oligonucleotide sequences of which are defined in theSequence Listing as follows:

Oligonucleotide SEQ ID NO. B3136CC 58 B3137CC 59 B3138CC 60 B3202CC 61

The resulting PCR fragments were gel-purified, restricted, and used toreplace the SpeI/BamHI and BamHI/SalI fragments in the V_(H) CDNA ofantibody CMX5-1.

CMX5-5 V_(L) DNA was constructed by replacing V_(L)3 (a BamHI/SalIfragment) of CMX5-1 in pSRS by a PCR-generated fragment using primersdesignated B3142CC and B3143CC (sequences defined by SEQ ID NO: 62 andSEQ ID NO: 63, respectively) and template CMX5-1 L.

The amino acid sequences of the heavy and light chain variable regionsof antibody CMX5-5 are defined in the Sequence Listing by SEQ ID NO: 64and SEQ ID NO: 65, respectively. Because amino acid residues 1-19 ofthese sequences comprise a secretory leader that is cleaved duringpost-translational processing, the actual variable region sequences aredefined by the sequences of SEQ ID NO: 64 and SEQ ID NO: 65, beginningwith residue 20.

Antibody Expression and Purification

To express the humanized antibodies, 5-10 μg each of pSRS-based L-chainplasmids and pSV.Sport-based H-chain plasmids were co-transfected into5×106 COS cells by electroporation protocols. The cells were then platedin a 60 mm culture dish in the presence of complete medium. After thecells settled and attached to the plates (about 6 hours aftertransfection and plating), the medium was aspirated and replaced withserum-free medium. Conditioned media were harvested at 72 hours.

The concentrations of humanized antibodies in conditioned media weredetermined using a human IgG4-specific enzyme-linked immunosorbent assay(ELISA). Nunc Immunoplates were coated at 4° C. for 24 hours with amouse anti-human IgG4-Fc monoclonal antibody (CalBiochem, La Jolla,Calif.) at 5 μg/ml in 50 mM bicarbonate buffer, pH 9.5. The plates werethen blocked at room temperature for 90 minutes with BLOTTO (5% non-fatdried milk and 0.05% (v/v) Tween-20 in Dulbecco's modified phosphatebuffered saline).

After washing away excess blocking reagent, serially diluted samples ina volume of 100 μl were applied to wells of the plates. The plates wereincubated at 37° C. for 2 hours, after which the samples were aspiratedand the wells were washed 3 times. One hundred microliters of sheepanti-human IgG (H+L) peroxidase conjugate (The Binding Site, San Diego,Calif.) were added to each well and the plates were incubated at 37° C.for 2 hours. The plates were then washed 3 times, and 100 μl of ABTSperoxidase substrate (Boehringer Mannheim, Indianapolis, Ind.) was addedto each well to produce a color reaction. The plates were readspectrophotometrically at 405 nm.

The conditioned media for all five of the humanized antibodies testedpositive for human IgG4. Repeated experiments showed that theconcentration of IgG4 was similar in the 3-day condition media forantibodies CMX5-1, CMX5-2, and CMX5-5 (about 200 μg per ml). The IgG4concentration of CMX5-4 conditioned media was usually about 2 to 3 foldhigher. IgG4 levels measured in conditioned medium containing antibodyCMX5-3, which contains the V_(H) framework from the LAY antibody insteadof the HIL antibody, were consistently about 5-10 fold lower than thelevels measured for antibodies CMX5-1, 2 and 5.

To obtain larger quantities of purified humanized antibodies,recombinant CHO cell lines were established that produced antibodyCMX5-1. CMX5-1 H- and L-chain plasmids pSRSMPA5H and pDSRGMPA5L wereco-transfected at a ratio of 20:1 into CHO cells, and stabletransfectants were selected for resistance to hypoxanthine and thymidinestarvation.

Of 106 resistant clones analyzed for human IgG4 secretion prior tomethotrexate (MTX) treatment, 65 clones tested positive. When the stableclones were subsequently subjected to methotrexate treatment to producegene amplification, the majority of the clones appeared to be highlyresistant to the drug. Although the cells were treated with MTX at 20 ,60 and 200 nM levels, only slight cell growth retardation was observedeven at the highest MTX concentration.

One of the better producer clones, designated CJA25, was continuouslytreated with MTX concentrations that were increased stepwise until afinal 1 mM concentration was reached. The final antibody expressionlevel at 1 mM MTX was estimated to be about 15 pg/cell/day. Thestability of this cell line was monitored by culturing the cells in theabsence of MTX for more than two months, during which the expressionlevel remained unchanged.

In a parallel experiment, CHO cells were again co-transfected withpDSRGMPASH and pSRSMPA5L but at a ratio of 1:20. The transfectionefficiency was found 10 to 100-fold lower, even though the conditionswere almost identical to those described above. Screening of 40 clonesshowed that 10 clones were positive for recombinant human IgG4secretion.

Levels of antibody JES1-39D10 antibody were measured in a similarfashion except that a goat anti-rat IgG Fc monoclonal antibody (Pierce,Rockford, Ill.) was used as the capture reagent and a sheep anti-rat IgG(H+L) peroxidase conjugate (The Binding Site, San Diego, Calif.) wasused as the detection reagent.

Antibodies in the conditioned media were purified by standard methodsusing protein G or protein A/G (Pierce Chemical) affinitychromatography, as described by the manufacturers of the chromatographicmaterials. The purified antibodies were more than 99% pure as determinedby amino acid composition analysis.

Antibodies in serum-free conditioned media from the JES1-39D10 hybridomaand from clone CJA25 were also purified by affinity chromatography usingcolumns containing immobilized human IL-5. These columns were preparedby coupling purified human IL-5 to AFFIGEL-151® resin (BioRad, Richmond,Calif.).

After loading conditioned medium from one of the sources, the column waswashed sequentially with phosphate buffered saline (PBS) and PBS plus0.5 M NaCl, and then re-equilibrated with PBS. Human IL-5-bindingantibodies were then eluted from the column with 0.2 M glycine at pH2.95, after which the eluted protein was immediately neutralized with 1M Tris-HCl, pH 8.

The concentration of antibodies thus purified was determined by UVabsorption at 280 nm, using determined molar extinction coefficients.The purified proteins were concentrated, dialyzed against phosphatebuffered saline, pH 7.2, and subjected to sodium dodecylsulfatepolyacrylamide gel electrophoresis [SDS-PAGE; Laemmli, Nature 227:680(1970)] in duplicate 10-20% gels under reducing conditions. After theelectrophoresis one gel was stained with Coomassie blue to visualize theprotein bands. The proteins in gels run in parallel were recovered byIMMOBILON® blotting, for amino-terminal sequence analyses.

Using this one-step purification method, the recovered protein wasestimated to be more than 99% pure, as determined by SDS-PAGE. Underreducing conditions, two bands were observed which had apparentmolecular weights of 50 and 23.2 kilodaltons, consistent with the knownmolecular weights of immunoglobulin H and L chains, respectively.

Antibody Characterization

Affinity Constants

To determine whether the humanized antibodies retained the ability tospecifically bind to human IL-5, apparent dissociation constants ofantibody/antigen complexes were measured. This was done by coatingenzyme immunoassay plates with mouse anti-human IgG₄-Fc (5 μg per ml,100 ill per well) in 50 mM bicarbonate buffer, pH 9.5. The plates werethen blocked at room temperature for 90 minutes with BLOTTO, washed 3times, and incubated at 37° C. for 2 hours with one of the humanizedantibodies, either purified (100 μl per well with a final concentrationof 0.05 μg per ml) or in the form of conditioned medium (100 μl perwell).

The recombinant antibody molecules from the conditioned media therebybecame immobilized on the plates through interactions between theconstant regions and the precoated antibodies so that the variableregions of the test antibodies were oriented in a uniform manner toallow direct and maximal interactions with the antigen.

To provide a standard for comparison, antibody JES1-39D10 was assayed inparallel, using goat anti-rat IgG Fc instead of the murine anti-humanIgG₄-Fc as the capture antibody.

After the plates were washed 3 times, a dilution series of ¹²⁵I-labeledhuman IL-5 at concentrations between 4,000 pM and 2 pM was applied tothe wells in each plate in final volumes of 100 μl. All test wells wererun in triplicate. Background binding was determined by using a1000-fold molar excess of unlablled human IL-5 in control wells. Thereactions were allowed to equilibrate at room temperature for 2 hours,at which time the wells were aspirated and washed 5 times with TBST. Thewells were then separated and counted in a Pharmacia LKB γ-counter. Allnonspecific binding controls were run in duplicate. The values obtainedfor bound human IL-5 were subjected to Scatchard analysis (using RADLIGsoftware) to obtain dissociation constant (kd) values.

Results from this analysis showed that the kd values obtained forhumanized antibodies CMX5-1, 2, 3 and 5 were all in the same approximaterange and were close to that of antibody JES1-39D10. The apparent kd ofCMX5-4 could not be determined because very little radioactivity wasdetected and no dose response was observed.

Competitive Binding Assays

To determine whether humanization introduced alterations in the antigenbinding region of antibody JES1-39D10, the wild-type antibody waslabeled with biotin using an X-NHS-biotin kit (Calbiochem, La Jolla,Calif.) with a final 20 mM reagent concentration. The product wasassayed in a direct binding ELISA to human IL-5, and a concentrationthat was about 3 times the apparent 50% point was used in competitionassays. This concentration corresponded to about 30 ng of biotinylatedantibody JES1-39D10.

Competition assays were carried out in which binding of the biotinylatedantibody to plates coated with human IL-5 was measured in the presenceof unlabelled antibody JES1-39D10 or antibody CMx5-1, CMX5-2 or CMX5-5,each of which had been purified by protein A/G affinity chromatography.

EIA plates were treated with 0.1 M NaHCO₃, pH 9.2, for 1 hour at roomtemperature. Human IL-5 in TBS was then added at 100 μg per well. Theplates were incubated overnight at 4° C. and then blocked with 3%BSA-TBS ml for 1 hour at room temperature. Fifty microliters of 2-foldserially diluted competing antibodies plus the appropriate amount ofbiotinylated antibody JES1-39D10 (also in a 50 μl volume) were added toeach well, and the plates were incubated for 1 hour at room temperature.

The plates were then washed 5 times with TBS-Tween-20, incubated with 1μg/ml horseradish peroxidase (HRP)-streptavidin conjugate for 1 hour atroom temperature, washed 5 times with TBS-Tween-20, and developed withHRP substrate TMB. Absorbance of the samples was then measuredspectrophotometrically at 450 nm.

The results showed that whereas unlabeled antibody JES1-39D10 competedwith its biotinylated form at an approximately 1:1 ratio, the samedegree of competition by antibodies CMX5-1, 2 and 5 required greateramounts of the antibodies. Taking the molar ratio of unlabeled tobiotinylated antibody JES1-39D10 to be 1.0, the molar ratios ofantibodies CMX5-1, 2 and 5 to biotinylated JES1-39D10 needed to producethe same degree of binding inhibition were estimated to be 3.3, 1.4 and1.4, respectively.

Inhibition of Human IL-5 Receptor Binding

In an investigation of the ability of humanized antibodies CMX5-1, 2 and5 to inhibit the binding of radiolabeled human IL-5 to recombinant humanIL-5 receptor α chains on transfected COS cells, it was found that allthree antibodies blocked receptor binding. This blocking activity wasobserved using both conditioned media and the purified antibodies. Witha constant 0.5 nM concentration of the labeled IL-5, the concentrationsof antibodies CMX5-1, 2 and 5 required to cause 50% inhibition ofreceptor binding (IC₅₀) were calculated to be 1.5-3.0, 0.35-0.7 and0.5-1.1 nM, respectively. The IC₅₀ for wild-type antibody JES1-39D10 wasdetermined to be 0.15-0.55 nM.

Modification of Antibody CMX5-5 Light Chain

All five of the humanized antibodies contained an aspartic acid residueat position 105 of the light chain due to DNA modifications made tofacilitate cloning. To restore the native sequence, the aspartic acidresidue at position 105 was replaced with a glutamic acid residue in thelight chain of antibody CMX5-5. This was done by using a syntheticoligonucleotide designated B3289CC (SEQ ID NO: 66) having an amino acidsequence corresponding to that of the junction of carboxyl-terminalpeptide of V_(L) and the amino-terminal peptide of the C_(L), with a GACto GAG codon change. This codon change created a XhoI restriction sitein place of an SalI site.

PCR was performed with oligonucleotide primers B3289CC and A2496CC (SEQID NO: 34), using the CMX5-5 light chain as the template. The resultingDNA fragment was gel-purified, restricted, and used to replace theSalI/EcoRI fragment in the pSRS-based CMX5-5 light chain cDNA. Themodified CMX5-5 light chain cDNA was then excised from pSRS and clonedinto pDSRG for stable expression. A new variant, consisting of theCMX5-2 heavy chain and the modified CMX5-5 light chain, was designatedCMX5-OK1. Another variant, consisting of the CMX5-5 heavy chain and themodified CMX5-5 light chain, was designated CMX5-OK2.

Biological Effects

Inhibition of IL-5-induced CD11b Expression

An HL-60 (ATCC CCL 240) subclone was maintained in Iscoves' ModifiedDulbecco's Medium (JRH BioSciences) supplemented with 10% FBS, 2mMglutamine, and penicillin-streptomycin. HL-60 is a multipotent humanpromyelocytic cell line which, in the presence of butyrate or humanIL-5, yields cells that develop the phenotypic characteristics ofeosinophils [Tomonaga et al., Blood 67:1433 (1986); Fabian et al., Blood80:788 (1992)]. Increased expression of a cell adhesion molecule calledCD11b/CD18 has been observed on the surface of eosinophils that wereactivated and recruited to the lung of allergic patients [Georas et al.,Am. J. Resp. Cell Mol. Biol. 7:261 (1992)].

Prior to assay, the HL-60 cells were primed for differentiation bygrowth in alkaline medium (pH adjusted to 7.6-7.8 with NaOH or sodiumbicarbonate) for one week. Following the period of growth, the cellswere transferred to 24-well culture dishes at a density of 2×105cells/ml. Serially diluted test antibodies was preincubated with aconstant amount of human IL-5 at 37° C. for one hour and then added tothe cells. The final concentration of IL-5 in the culture was 150 pM.

Seventy-two hours later, the cells were harvested by centrifugation andresuspended at a concentration of 1×10⁶ cells/ml and 50 μl volumes(5×10⁴ cells) were aliquoted per well in a 96-well microtiter plate. Thecells were dried in the wells and then washed with 70% ethanol followedby TBS-Tween-20, and blocked with 10% non-fat dried milk and 5% BSA.

The primary antibody (mouse anti-human CD11b; Becton-Dickinson,Braintree, Mass.) was added. After a two-hour incubation at 37° C., theplates were treated sequentially by washing 3× with TBS-tween-20,allowing a secondary antibody against the primary antibody (JacksonImmunoResearch Laboratories, West Grove, Pa.) to bind, and washing again3×with TBS-Tween-20. Specifically bound antibody was then detected usingan ELISA Amplification System (Gibco-BRL).

It was found that humanized antibodies CMX5-1, CMX5-2 and CMX5-5 wereall able to block the CD11b induction activity of human IL-5. Therelative IC₅₀'s were estimated to be as follows:

IC₅₀ MOLAR RATIO SAMPLE (pM) CMX5/IL-5 IC₅₀ CMX5/IC₅₀ 39D10 CMX5-1 1467 7.3:1 8.8 CMX5-2  467  2.3:1 2.8 CMX5-5  800   4:1 4.8 39D10  1660.83:1 1  

As can be seen from the Table, humanized antibodies CMX5-2 and CMX5-5appeared to be more potent than antibody CMX5-1.

Inhibition of Allergen-induced Mouse Eosinophilia

To determine whether the humanized antibodies were capable ofneutralizing the activity of IL-5 in vivo, young male B6D2F1/J mice weresensitized with alum-precipitated ovalbumin, using 8 mg per animal. Oneweek thereafter a booster dose was given. Except for a group of 5unsensitized control mice, all other groups contained 6 mice.

One week after the booster and prior to challenge, the sensitizedanimals were injected intraperitoneally with test antibodies in a volumeof 0.5 ml. Each group received one of the following: 0.1 mg of antibodyJES1-39D10 per kilogram of body weight, 1 mg of antibody JES1-39D10 perkilogram of body weight, 1 mg of antibody CMX5-1 per kilogram of bodyweight, and 10 mg of antibody CMX5-1 per kilogram of body weight. Anantibody designated TRFK 5, a rat anti-mouse IL-5 monoclonal antibody(Schumacher et al., supra), was used as a positive control. The controlsensitized mice received saline.

The animals were then challenged twice with ovalbumin aerosol for 1hour. Twenty-four hours following the challenge, bronchioalveolarlavage, peripheral blood, and lung-tissue specimens were collected,fixed, developed by eosinophil-specific staining dyes, and examinedmicroscopically to determine eosinophil distribution.

Mice that received antibody CMX5-1 at the 10 mg per kilogram body ofweight dose were found to have significantly reduced numbers ofeosinophils in their bronchioalveolar lavage fluid, whereas lessquantitative changes were observed with other cell types. Mice thatreceived antibody JES1-39D10 also had reduced eosinophil levels.

Hybridoma Deposit

The hybridoma cell line (JES1-39D10.11) producing monoclonal antibodyJES1-39D10 was deposited Jan. 8, 1992 with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209,and assigned Accession No. ATCC HB 10959. These deposits were made underconditions as provided under ATCC's agreement for Culture Deposit forPatent Purposes, which assures that the deposits will be made availableto the US Commissioner of Patents and Trademarks pursuant to 35 USC 122and 37 CFR 1.14 and will be made available to the public upon issue of aU.S. patent, and which requires that the deposits be maintained.Availability of the deposited strains is not to be construed as alicense to practise the invention in contravention of the rights grantedunder the authority of any government in accordance with its patentlaws.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will become apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims.

68 1 333 DNA Mus musculus 1 gaatctggag gaggcttggt acagccatca cagaccctgtctctcacctg cactgtctct 60 gggttatcat taaccagcaa tagtgtgaac tggattcggcagcctccagg aaagggtctg 120 gagtggatgg gactaatatg gagtaatgga gacacagattataattcagc tatcaaatcc 180 cgactgagca tcagtaggga cacctcgaag agccaggttttcttaaagat gaacagtctg 240 caaagtgaag acacagccat gtacttctgt gccagagagtactacggcta ctttgattac 300 tggggccaag gagtcatggt cacagtctcc tca 333 2 384DNA Mus musculus 2 atggctgtgc ccactcagct cctggggttg ttgttgctgtggattacaga tgccatatgt 60 gacatccaga tgacacagtc tccagcttcc ctgtctgcatctctgggaga aactatctcc 120 atcgaatgtc tagcaagtga gggcatttcc agttatttagcgtggtatca gcagaagcca 180 gggaaatctc ctcagctcct gatctatggt gcaaatagcttgcaaactgg ggtcccatca 240 cggttcagtg gcagtggatc tgccacacaa tattctctcaagatcagcag catgcaacct 300 gaagatgaag gggattattt ctgtcaacag agttacaagtttccgaacac gtttggagct 360 gggaccaagc tggaactgaa acgg 384 3 39 DNA Rattusrattus 3 agtctagaag cttgaatctg gaggaggctt ggtacagcc 39 4 58 DNA Rattusrattus 4 cagcccggga attcgtcgac tcactgccat gtttctttct ttacattgag cttgctgt58 5 36 DNA Rattus rattus 5 gcaagcttgg atccagacag gacacaggcc agacat 36 638 DNA Rattus rattus 6 cacgaattct gcagtggcac ctcaggacct ttgggtct 38 7114 DNA Rattus rattus 7 aggcagtcga cgccgccacc atgaagttgt ggctgaactggattttcctt ttaacacttt 60 taaatggtat ccagtgtgag gtgaaactgt tggaatctggaggaggcttg gtac 114 8 131 DNA Rattus rattus 8 actgaattct atttaccaggagagtgggag agactcttct cagtatggtg gttgtgcagg 60 ccctcatgca gcacagaacacgtgaaagtg tttccctgct gccatgtttc tttctttaca 120 ttgagcttgc t 131 9 66DNA Rattus rattus 9 agctgtcgac gccgccacca tgcgttgtgc cactcagctcctggggttgt tgttgctgtg 60 gattac 66 10 48 DNA Rattus rattus 10 agctctagaattctgcagtc aacactcatt cctgttgaag ctcttgac 48 11 102 DNA ArtificialSequence This antibody contains mouse and human sequences 11 gctgaattcgccgccaccat gggctggagc tgtatcatcc tcttcttagt agcaacagct 60 acaggtgtccactcccaggt caaactggta caagctggag gt 102 12 102 DNA Artificial SequenceThis antibody contains mouse and human sequences 12 gcgtactagttaatgataac ccagagacga tgcaactcag tcgcagagat cttcctggct 60 gtacgacgccacctccagct tgtaccagtt tgacctggga gt 102 13 86 DNA Artificial SequenceThis antibody contains mouse and human sequences 13 gtcagactagtaatagtgtg aactggatac ggcaagcacc tggcaagggt ctggagtggg 60 ttgcactaatatggagtaat ggagac 86 14 73 DNA Artificial Sequence This antibodycontains mouse and human sequences 14 gtactctaga gattgtgaat cgagatttgatagctgaatt ataatctgtg tctccattac 60 tccatattag tgc 73 15 104 DNAArtificial Sequence This antibody contains mouse and human sequences 15gcagaattct agagacaatt cgaagagcac cctatacatg cagatgaaca gtctgagaac 60tgaagatact gcagtctact tctgtgctcg tgagtactat ggat 104 16 99 DNAArtificial Sequence This antibody contains mouse and human sequences 16ctcgtgagct cgggcccttg gtcgacgctg aggagactgt gactaggaca ccttgacccc 60aatagtcgaa atatccatag tactcacgag cacagaagt 99 17 94 DNA ArtificialSequence This antibody contains mouse and human sequences. 17 agagctgcagccgccaccat gggatggagc tgtatcatcc tcttcttggt agcaacagct 60 acaggtgtccactccgacat ccagatgaca cagt 94 18 83 DNA Artificial Sequence Thisantibody contains mouse and human sequences. 18 agcatctaga ggtgaccctatctccgacag atacagacag cgaacttgga gactgtgtca 60 tctggatgtc ggagtggaca cct83 19 101 DNA Artificial Sequence This antibody contains mouse and humansequences. 19 agatctgcag gtcaccatca catgtctagc aagtgagggc atctccagttacttagcgtg 60 gtaccagcag aagcccgggc tagctcctaa gctcctgatc t 101 20 84DNA Artificial Sequence This antibody contains mouse and humansequences. 20 atggcggatc ctgagccact gaatcttgat ggtactccag tctgcaagctattcgcacca 60 tagatcagga gcttaggagc tagc 84 21 81 DNA ArtificialSequence This antibody contains mouse and human sequences. 21 ctcaggatccgctacagact tcacgctcac gatctccagc ctacagcctg aagatatcgc 60 gacgtattactgtcaacagt c 81 22 76 DNA Artificial Sequence This antibody containsmouse and human sequences. 22 gcatgccgtc gaccttggtg ccttgaccgaatgtgttcgg gaacttatac gactgttgac 60 agtaatacgt cgcgat 76 23 70 DNA Homosapiens 23 gcatcgcgtc gaccaaaggt ccatctgtgt ttccgctggc gccatgctccaggagcacct 60 ccgagagcac 70 24 79 DNA Homo sapiens 24 gacagaattcaggtgctgga cacgacggac atggaggacc atacttcgac tcaactctct 60 tgtccaccttggtgttgct 79 25 68 DNA Homo sapiens 25 actggaattc ctaggtggac catcagtcttcctgtttccg cctaagccca aggacactct 60 catgatct 68 26 51 DNA Homo sapiens26 caggctgtcg actcgaggct gacctttggc tttggagatg gttttctcga t 51 27 38 DNAHomo sapiens 27 gtaagcgtcg actcgagagc cacaggtgta caccctgc 38 28 40 DNAHomo sapiens 28 cgctagcggc cgctcattta cccagagaca gggagaggct 40 29 196DNA Homo sapiens 29 agtgcgctgc agccgccacc atgggatgga gctgtatcatcctcttcttg gtagcaacag 60 ctacaggtgt ccactccgac atccagatga cacagtctccaagttccctg tctgcatctg 120 tcggagatcg ggtcacaatc gaatgtctag caagtgagggcatttccagt tatttagcgt 180 ggtatcagca gaagcc 196 30 213 DNA Homo sapiens30 accttggtac cttgtccaaa cgtgttcgga aacttgtaac tctgttgaca gtaataatct 60ccttcatctt caggttgcag gctggagatc ttaaacgtga aatctgtgcc ggatccactg 120ccactgaacc gtgatgggac cccagtttgc aagctatttg caccatagat caggagttta 180ggagctttcc ctggcttctg ctgataccac gct 213 31 190 DNA Homo sapiens 31gaacacgttt ggacaaggta ccaaggtcga catcaaacgg actgtggctg caccatctgt 60cttcatcttc ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct 120gctgaataac ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca 180atcgggtaac 190 32 207 DNA Homo sapiens 32 gtcagaattc taacactctcccctgttgaa gctctttgtg acgggcgagc tcaggccctg 60 atgggtgact tcgcaggcgtagactttgtg tttctcgtag tctgctttgc tcagcgtcag 120 ggtgctgctg aggctgtaggtgctgtcctt gctgtcctgc tctgtgacac tctcctggga 180 gttacccgat tggagggcgttatccac 207 33 32 DNA Homo sapiens 33 gcatgcgtcg acgtcaaacg gactgtggctgc 32 34 111 DNA Homo sapiens 34 gatcaagctt gaattctaac actctcctctgttgaagctc ttcgtgactg gcgagctcag 60 gccttgatga gtgacttcgc aggcgtagactttgtgtttc tcgtagtctg c 111 35 117 DNA Homo sapiens 35 gagtcagtccaagcttgaat tctaacactc tcctctgttg aagctcttcg tgactggcga 60 gctcaggccttgatgagtga cttcgcaggc gtagactttg tgtttctcgt agtctgc 117 36 135 PRTArtificial Sequence This antibody contains both human and mousesequences. 36 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr AlaThr Gly 1 5 10 15 Val His Ser Gln Val Lys Leu Val Gln Ala Gly Gly GlyVal Val Gln 20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ile Val Ser GlyLeu Ser Leu 35 40 45 Thr Ser Asn Ser Val Asn Trp Ile Arg Gln Ala Pro GlyLys Gly Leu 50 55 60 Glu Trp Val Ala Leu Ile Trp Ser Asn Gly Asp Thr AspTyr Asn Ser 65 70 75 80 Ala Ile Lys Ser Arg Phe Thr Ile Ser Arg Asp AsnSer Lys Ser Thr 85 90 95 Leu Tyr Met Gln Met Asn Ser Leu Arg Thr Glu AspThr Ala Val Tyr 100 105 110 Phe Cys Ala Arg Glu Tyr Tyr Gly Tyr Phe AspTyr Trp Gly Gln Gly 115 120 125 Val Leu Val Thr Val Ser Ser 130 135 37127 PRT Artificial Sequence The antibody contains human and mousesequences. 37 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr AlaThr Gly 1 5 10 15 Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser SerLeu Ser Val 20 25 30 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Leu Ala SerGlu Gly Ile 35 40 45 Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly LeuAla Pro Leu 50 55 60 Leu Leu Ile Tyr Gly Ala Asn Ser Leu Gln Thr Gly ValPro Ser Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Ala Thr Asp Phe Thr LeuThr Ile Ser Ser 85 90 95 Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys GlnGln Ser Tyr Lys 100 105 110 Phe Pro Asn Thr Phe Gly Gln Gly Thr Lys ValAsp Val Lys Arg 115 120 125 38 42 DNA Artificial Sequence This antibodycontains human and mouse sequences 38 gatctctgcg actgagttgc atcgcatctgggttcacatt ct 42 39 42 DNA Artificial Sequence This antibody containshuman and mouse sequences. 39 gatctctgcg actgagttgc atcgcatctgggttcacatt ct 42 40 135 PRT Artificial Sequence Mouse and human 40 MetGly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15Val His Ser Gln Val Lys Leu Val Gln Ala Gly Gly Gly Val Val Gln 20 25 30Pro Gly Arg Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly Phe Thr Phe 35 40 45Ser Ser Asn Ser Val Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ala Leu Ile Trp Ser Asn Gly Asp Thr Asp Tyr Asn Ser 65 70 7580 Ala Ile Lys Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Thr 85 9095 Leu Tyr Met Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Val Tyr 100105 110 Phe Cys Ala Arg Glu Tyr Tyr Gly Tyr Phe Asp Tyr Trp Gly Gln Gly115 120 125 Val Leu Val Thr Val Ser Ser 130 135 41 127 PRT ArtificialSequence Mouse and human 41 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu ValAla Thr Ala Thr Gly 1 5 10 15 Val His Ser Asp Ile Gln Met Thr Gln SerPro Ser Ser Leu Ser Val 20 25 30 Ser Val Gly Asp Arg Val Thr Ile Thr CysLeu Ala Ser Glu Gly Ile 35 40 45 Ser Ser Tyr Leu Ala Trp Tyr Gln Gln LysPro Gly Leu Ala Pro Lys 50 55 60 Leu Leu Ile Tyr Gly Ala Asn Ser Leu GlnThr Gly Val Pro Ser Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Ala Thr AspPhe Thr Leu Thr Ile Ser Ser 85 90 95 Leu Gln Pro Glu Asp Ile Ala Thr TyrTyr Cys Gln Gln Ser Tyr Lys 100 105 110 Phe Pro Asn Thr Phe Gly Gln GlyThr Lys Val Asp Val Lys Arg 115 120 125 42 95 DNA Homo sapiens 42gcagctgcag ccgccaccat gggctggagc tgtatcatcc tcttcttagt agcaacagct 60acaggtgtcc actccgaggt ccagctgcta gagtc 95 43 108 DNA Homo sapiens 43agacgaattc actagttaat gataacccag agactgcgca actcagtcgc agagatcctc 60ctggctgtac gaggccacct ccagactcta gcagctggac ctcggagt 108 44 103 DNA Homosapiens 44 aagcgatcta gaaatgactc gaagaacacc ctatacctac agatgaacggtctgcaagct 60 gaagtaagtg caatctactt ctgtgctcgt gagtactatg gat 103 45 93DNA Homo sapiens 45 acgagaagct tcatgtcgac gctgaggaga ctgtgactagcgtaccttga ccccaatagt 60 cgaaatatcc atagtactca cgagcacaga agt 93 46 135PRT Artificial Sequence This antibody contains mouse and humansequences. 46 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr AlaThr Gly 1 5 10 15 Val His Ser Glu Val Gln Leu Leu Glu Ser Gly Gly GlyLeu Val Gln 20 25 30 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser GlyLeu Ser Leu 35 40 45 Thr Ser Asn Ser Val Asn Trp Ile Arg Gln Ala Pro GlyLys Gly Leu 50 55 60 Glu Trp Val Ala Leu Ile Trp Ser Asn Gly Asp Thr AspTyr Asn Ser 65 70 75 80 Ala Ile Lys Ser Arg Phe Thr Ile Ser Arg Asn AspSer Lys Asn Thr 85 90 95 Leu Tyr Leu Gln Met Asn Gly Leu Gln Ala Glu ValSer Ala Ile Tyr 100 105 110 Phe Cys Ala Arg Glu Tyr Tyr Gly Tyr Phe AspTyr Trp Gly Gln Gly 115 120 125 Thr Leu Val Thr Val Ser Ser 130 135 47127 PRT Artificial Sequence This antibody contains mouse and humansequences. 47 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr AlaThr Gly 1 5 10 15 Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser SerLeu Ser Val 20 25 30 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Leu Ala SerGlu Gly Ile 35 40 45 Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly LeuAla Pro Lys 50 55 60 Leu Leu Ile Tyr Gly Ala Asn Ser Leu Gln Thr Gly ValPro Ser Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Ala Thr Asp Phe Thr LeuThr Ile Ser Ser 85 90 95 Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys GlnGln Ser Tyr Lys 100 105 110 Phe Pro Asn Thr Phe Gly Gln Gly Thr Lys ValAsp Val Lys Arg 115 120 125 48 97 DNA Artificial Sequence Mouse andhuman 48 gctgactgca gccgccacca tgggctggag ctgtatcatc ctcttcttagtagcaacagc 60 tacaggtgtc cactcccagg tcaaactggt acaagct 97 49 108 DNAArtificial Sequence Mouse and human 49 ctagaagctt actagttaat gataacccagatgcgatgca actcagtcgc agagatcttc 60 ctggctgtac gacgccacct ccagcttgtaccagtttgac ctgggagt 108 50 90 DNA Artificial Sequence Mouse and human 50ggacgaattc actagtaatg gtatgcactg ggtacggcaa gcacctggca agggtctgga 60gtgggttgca gtaatatgga gtaatggatc 90 51 85 DNA Artificial Sequence Mouseand human 51 actgctctag agattgtgaa tcgtcctttg actgagtcac catagtatgttcgtgatcca 60 ttactccata ttactgcaac ccact 85 52 95 DNA ArtificialSequence Mouse and human 52 cgtactctag agacaattcg aagcgcaccc tatacatgcagatgaacagt ctgagaactg 60 aagatactgc tgtctactac tgtgctcgtg agtac 95 53100 DNA Artificial Sequence Mouse and human 53 acgagaagct tcatgtcgacgctgaggaga ctgtgactag gacaccttga ccccaatagt 60 cgaaatatcc atagtactcacgagcacagt agtagacagc 100 54 153 DNA Artificial Sequence Mouse and human54 gcgataggtc accatcacat gtcaagcaag tgagggcatc tccagttact taaactggta 60tcagcagaag cccgggctag ctcctaagct cctgatctat ggtgcgaata ccagggaggc 120tggagtacca tcaagattca gtggctcagg ctc 153 55 150 DNA Artificial SequenceMouse and human 55 acagtcgtcg accttggtgc cttgaccgaa tgtgttcgggaacttatacg actgttgaca 60 gtaatacgtc gcgatatctt caggctgtag gctggagatcgtgagcgtga agtctgtacc 120 ggagcctgag ccactgaatc ttgatggtac 150 56 136PRT Artificial Sequence This sequence contains mouse and humansequences. 56 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr AlaThr Gly 1 5 10 15 Val His Ser Gln Val Lys Leu Val Gln Ala Gly Gly GlyVal Val Gln 20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ile Ala Ser GlyLeu Ser Leu 35 40 45 Thr Ser Asn Gly Met His Trp Val Arg Gln Ala Pro GlyLys Gly Leu 50 55 60 Glu Trp Val Ala Val Ile Trp Ser Asn Gly Ser Arg ThrTyr Tyr Gly 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg AspAsn Ser Lys Arg 85 90 95 Thr Leu Tyr Met Gln Met Asn Ser Leu Arg Thr GluAsp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Glu Tyr Tyr Gly Tyr PheAsp Tyr Trp Gly Gln 115 120 125 Gly Val Leu Val Thr Val Ser Ser 130 13557 127 PRT Artificial Sequence This sequence contains mouse and humansequences. 57 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr AlaThr Gly 1 5 10 15 Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser SerLeu Ser Val 20 25 30 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ala SerGlu Gly Ile 35 40 45 Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly LeuAla Pro Lys 50 55 60 Leu Leu Ile Tyr Gly Ala Asn Thr Arg Glu Ala Gly ValPro Ser Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr LeuThr Ile Ser Ser 85 90 95 Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys GlnGln Ser Tyr Lys 100 105 110 Phe Pro Asn Thr Phe Gly Gln Gly Thr Lys ValAsp Val Lys Arg 115 120 125 58 98 DNA Artificial Sequence This sequencecontains mouse and human sequences. 58 gcatgacagt agatctctgc gactgagttgcatcgcatct gggttcacat tctctagtaa 60 tagtgtgaac tgggtacggc aagcacctggcaagggtc 98 59 113 DNA Artificial Sequence This sequence contains mouseand human sequences. 59 acgatcactc tagagattgt gaatcgagat ttgatagctgaattataatc tgtgtctcca 60 ttactccata ttagtgcaac ccactccaga cccttgccaggtgcttgccg tac 113 60 91 DNA Artificial Sequence This sequence containsmouse and human sequences. 60 gcatggacgt ctagagacaa ttcgaagagaaccctataca tgcagatgaa cagtctgaga 60 actgaagata ctgcagtcta ctactgtgct c91 61 120 DNA Artificial Sequence This sequence contains mouse and humansequences. 61 caagtcgacg acaagcttgt cgacgctgag gagactgtga ctaggacaccttgaccccaa 60 tagtcgaaat atccatagta ctcacgagca cagtagtaga ctgcagtatcttcagttctc 120 62 28 DNA Artificial Sequence This sequence containsmouse and human sequences. 62 acagtccgtt tgacgtcgac cttggtgc 28 63 63DNA Artificial Sequence This sequence contains mouse and humansequences. 63 agtggctcag gatccggtac cgacttcacg ttcacgatct ccagcctacagcctgaagat 60 atc 63 64 135 PRT Artificial Sequence This sequencecontains mouse and human sequences. 64 Met Gly Trp Ser Cys Ile Ile LeuPhe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Lys LeuVal Gln Ala Gly Gly Gly Val Val Gln 20 25 30 Pro Gly Arg Ser Leu Arg LeuSer Cys Ile Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Asn Ser Val Asn TrpVal Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala Leu Ile TrpSer Asn Gly Asp Thr Asp Tyr Asn Ser 65 70 75 80 Ala Ile Lys Ser Arg PheThr Ile Ser Arg Asp Asn Ser Lys Arg Thr 85 90 95 Leu Tyr Met Gln Met AsnSer Leu Arg Thr Glu Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Ala Arg GluTyr Tyr Gly Tyr Phe Asp Tyr Trp Gly Gln Gly 115 120 125 Val Leu Val ThrVal Ser Ser 130 135 65 127 PRT Artificial Sequence This sequencecontains mouse and human sequences 65 Met Gly Trp Ser Cys Ile Ile LeuPhe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Asp Ile Gln MetThr Gln Ser Pro Ser Ser Leu Ser Val 20 25 30 Ser Val Gly Asp Arg Val ThrIle Thr Cys Leu Ala Ser Glu Gly Ile 35 40 45 Ser Ser Tyr Leu Ala Trp TyrGln Gln Lys Pro Gly Leu Ala Pro Lys 50 55 60 Leu Leu Ile Tyr Gly Ala AsnSer Leu Gln Thr Gly Val Pro Ser Arg 65 70 75 80 Phe Ser Gly Ser Gly SerGly Thr Asp Phe Thr Phe Thr Ile Ser Ser 85 90 95 Leu Gln Pro Glu Asp IleAla Thr Tyr Tyr Cys Gln Gln Ser Tyr Lys 100 105 110 Phe Pro Asn Thr PheGly Gln Gly Thr Lys Val Asp Val Lys Arg 115 120 125 66 40 DNA ArtificialSequence This sequence contains mouse and human sequences. 66 agcgagcgctcgaggtcaaa cggactgtgg ctgcaccatc 40 67 111 PRT Artificial Sequence Thisantibody contains mouse and human sequences 67 Glu Ser Gly Gly Gly LeuVal Gln Pro Ser Gln Thr Leu Ser Leu Thr 1 5 10 15 Cys Thr Val Ser GlyLeu Ser Leu Thr Ser Asn Ser Val Asn Trp Ile 20 25 30 Arg Gln Pro Pro GlyLys Gly Leu Glu Trp Met Gly Leu Ile Trp Ser 35 40 45 Asn Gly Asp Thr AspTyr Asn Ser Ala Ile Lys Ser Arg Leu Ser Ile 50 55 60 Ser Arg Asp Thr SerLys Ser Gln Val Phe Leu Lys Met Asn Ser Leu 65 70 75 80 Gln Ser Glu AspThr Ala Met Tyr Phe Cys Ala Arg Glu Tyr Tyr Gly 85 90 95 Tyr Phe Asp TyrTrp Gly Gln Gly Val Met Val Thr Val Ser Ser 100 105 110 68 128 PRTArtificial Sequence This antibody contains mouse and human sequences 68Met Ala Val Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp Ile Thr 1 5 1015 Asp Ala Ile Cys Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser 20 2530 Ala Ser Leu Gly Glu Thr Ile Ser Ile Glu Cys Leu Ala Ser Glu Gly 35 4045 Ile Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro 50 5560 Gln Leu Leu Ile Tyr Gly Ala Asn Ser Leu Gln Thr Gly Val Pro Ser 65 7075 80 Arg Phe Ser Gly Ser Gly Ser Ala Thr Gln Tyr Ser Leu Lys Ile Ser 8590 95 Ser Met Gln Pro Glu Asp Glu Gly Asp Tyr Phe Cys Gln Gln Ser Tyr100 105 110 Lys Phe Pro Asn Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu LysArg 115 120 125

What is claimed is:
 1. A polypeptide which specifically binds humaninterleukin-5, comprising amino acid residues 21-27, 47-50, 66 and93-101 of SEQ ID NO:67 grafted into a human heavy chain variable region(V_(H)) sequence framework and amino acid residues 46-51, 68, 70-72, 84,91, 109 and 110-116 of SEQ ID NO:68 grafted into a human light chainvariable region (V_(L)) sequence framework.
 2. A polypeptide whichspecifically binds human interleukin-5, comprising amino acid residues26-30, 45-60 and 93-100 of SEQ ID NO:67 grafted into a human heavy chainvariable region (V_(H)) sequence framework and amino acid residues44-54, 70-76 and 109-117 of SEQ ID NO:68 grafted into a human lightchain variable region (V_(L)) sequence framework.
 3. The polypeptide ofeither claim 1 or claim 2 in which the human V_(H) sequence frameworkand the human V_(L) sequence framework are from the same human antibody.4. The polypeptide of claim 3 in which the human antibody is the HILantibody.
 5. The polypeptide of claim 3 in which the human antibody isthe LAY antibody.
 6. The polypeptide of either claim 1 or claim 2 inwhich the human V_(H) sequence framework and the human V_(L) sequenceframework are from different human antibodies.
 7. The polypeptide ofclaim 6 in which the human V_(H) sequence framework is from the humanantibody HIL and the human V_(L) sequence framework is from the humanantibody LAY.
 8. The polypeptide of claim 6 in which the human V_(H)sequence framework is from the human antibody LAY and the human V_(L)sequence framework is from the human antibody HIL.
 9. The polypeptide ofeither claim 1 or claim 2 further comprising a human antibody _(γ)4constant region.
 10. The polypeptide of claim 1 comprising SEQ ID NO:36and SEQ ID NO:37.
 11. The polypeptide of claim 1 comprising SEQ ID NO:40and SEQ ID NO:41.
 12. The polypeptide of claim 1 comprising SEQ ID NO:46and SEQ ID NO:47.
 13. The polypeptide of claim 1 comprising SEQ ID NO:56and SEQ ID NO:57.
 14. The polypeptide of claim 2 comprising SEQ ID NO:64and SEQ ID NO:65.
 15. A monoclonal antibody or fragment thereof whichspecifically binds human interleukin-5 comprising: (a) a heavy chainvariable region defined by a sequence selected from the group consistingof SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:46, SEQ ID NO:56, and SEQ IDNO:64; and (b) a light chain variable region defined by a sequenceselected from the group consisting of SEQ ID NO:37, SEQ ID NO: 41, SEQID NO:47, SEQ ID NO:57, and SEQ ID NO:65.
 16. A method of making ahumanized monoclonal antibody or fragment thereof which specificallybinds to human interleukin-5 and which comprises complementaritydetermining regions (CDRs) from the heavy chain variable region (V_(H))sequence defined by SEQ ID NO: 1 and/or the light chain variable region(V_(L)) sequence defined by SEQ ID NO:2, said method comprising: (a)cloning cDNAs encoding CDRs from the V_(H) sequence defined by SEQ IDNO:1 and/or the V_(L) sequence defined by SEQ ID NO:2; (b) selecting ahuman antibody sequence to be used as a human framework for the V_(H)sequence and/or the V_(L) sequence of step (a); (c) grafting the cDNAsencoding CDRs from the V_(H) sequence of step (a) into a polynucleotidesequence encoding the human framework V_(H) sequence of step (b) and/orgrafting the cDNAs encoding CDRs from the V_(L) sequence of step (a)into a polynucleotide sequence encoding the human framework V_(L)sequence of step (b), thereby creating a polynucleotide encoding ahumanized monoclonal antibody or fragment thereof; (d) cloning thepolynucleotide encoding the humanized monoclonal antibody or fragmentthereof from step (c) into an expression vector; (e) transfecting theexpression vector into a host cell; (f) culturing a host cell comprisingthe expression vector of step (d) under conditions in which thepolynucleotide encoding the humanized monoclonal antibody or fragmentthereof is expressed; and (g) recovering the expressed humanizedmonoclonal antibody or fragment thereof.
 17. The method of claim 16 inwhich the human antibody sequence is selected by a method comprising:(a) comparing the heavy chain variable region (V_(H)) and light chainvariable region (V_(L)) sequences of an animal monoclonal antibody thatis to be humanized with optimally-aligned sequences of the V_(H) andV_(L) of all human antibodies for which sequence information isavailable, thereby determining the percent identity for each comparedhuman V_(H) and V_(L) sequence; (b) determining the number ofambiguities in each human antibody V_(H) and V_(L) sequence of step (a)as compared to the respective V_(H) and V_(L) sequence of the animalmonoclonal antibody that is to be humanized; (c) comparing Pin-regionspacing of the animal monoclonal antibody V_(H) and V_(L) sequences ofstep (a) with (i) the Pin-region spacing of each human antibody V_(H)and V_(L) sequence compared in step (a) and with (ii) the Pin-regionspacing of other antibodies which have known 3-dimensional structures;and (d) selecting the human antibody which has the best combination of(i) high percent identity as determined in step (a), (ii) low number ofsequence ambiguities as determined in step (b) and (iii) similarPin-region spacing as determined in step (c).